Compositions and methods for delivery of nucleic acids to cells

ABSTRACT

Compositions and methods of use thereof for delivering nucleic acid cargo into cells are provided. The compositions typically include (a) a 3E10 monoclonal antibody or an antigen binding, cell-penetrating fragment thereof; a monovalent, divalent, or multivalent single chain variable fragment (scFv); or a diabody; or humanized form or variant thereof, and (b) a nucleic acid cargo including, for example, a nucleic acid encoding a polypeptide, a functional nucleic acid, a nucleic acid encoding a functional nucleic acid, or a combination thereof. Elements (a) and (b) are typically non-covalently linked to form a complex.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalApplication No. 62/944,281, entitled “Compositions And Methods ForDelivery Of Nucleic Acids To Cells”, filed in the United States Patentand Trademark Office on Dec. 5, 2019, International Application No.PCT/US2019/048953, entitled “Compositions And Methods For EnhancingDonor Oligonucleotide-Based Gene Editing” and filed in the United StatesReceiving Office for the Patent Cooperation Treaty on Aug. 30, 2019, andInternational Application No. PCT/US2019/048962, entitled “CompositionsAnd Methods For Enhancing Triplex And Nuclease-Based Gene Editing” andfiled in the United States Receiving Office for the Patent CooperationTreaty on Aug. 30, 2019. U.S. Provisional Application No. 62/944,281,International Application No. PCT/US2019/048953, U.S. ProvisionalApplication No. 62/725,920, International Application No.PCT/US2019/048962, U.S. Provisional Application No. 62/725,852 are eachspecifically incorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under CA197574 awardedby National Institutes of Health. The Government has certain rights inthe invention.

REFERENCE TO THE SEQUENCE LISTING

The Sequence Listing submitted as a text file named “YU_7503_3_ST25”created on Aug. 31, 2020, and having a size of 154,701 bytes is herebyincorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

FIELD OF THE INVENTION

The invention is generally related to the field of intracellulardelivery of nucleic acids, for application including, but not limited toin vitro, ex vivo, and in vivo gene therapy and gene editing.

BACKGROUND OF THE INVENTION

Gene therapy includes a spectrum of applications ranging from genereplacement and knockdown for genetic or acquired diseases such ascancer, to vaccination. Viral vectors and synthetic liposomes haveemerged as the vehicles of choice for many applications today, but bothhave limitations and risks, including complexity of production, limitedpackaging capacity, and unfavorable immunological features, whichrestrict gene therapy applications and hold back the potential forpreventive gene therapy (Seow and Wood, Mol Ther. 17(5): 767-777 (2009).

In vivo uptake and distributed of nucleotide in cells and tissues hasbeen observed (Huang, et al., FEBS Lett., 558(1-3):69-73 (2004)).Further, although, for example, Nyce, et al. have shown that antisenseoligodeoxynucleotides (ODNs) when inhaled bind to endogenous surfactant(a lipid produced by lung cells) and are taken up by lung cells withouta need for additional carrier lipids (Nyce, et al., Nature, 385:721-725(1997)), small nucleic acids are taken up into T24 bladder carcinomatissue culture cells (Ma, et al., Antisense Nucleic Acid Drug Dev.,8:415-426 (1998)), there remains a need for improved nucleic acidtransfection technology, particularly for in vivo applications. AAV9,still the viral vector typically used in people was discovered in 2003(Robbins, “Gene therapy pioneer says the field is behind—and thatdelivery technology is embarrassing,” Stat, November, 2019).

Thus, it is an object of the invention to provided compositions andmethods of use thereof for improved delivery of nucleic acids intocells.

SUMMARY OF THE INVENTION

Compositions and methods of use thereof for delivering nucleic acidcargo into cells are provided. The compositions typically include (a) a3E10 monoclonal antibody or a cell-penetrating fragment thereof; amonovalent, divalent, or multivalent single chain variable fragment(scFv); or a diabody; or humanized form or variant thereof, and (b) anucleic acid cargo including, for example, a nucleic acid encoding apolypeptide, a functional nucleic acid, a nucleic acid encoding afunctional nucleic acid, or a combination thereof. Elements (a) and (b)are typically non-covalently linked to form a complex. It is believedthat in additional to DNA, 3E10 binds to RNA, PNA, and other nucleicacids.

Exemplary 3E10 antibodies and fragments and fusion protein thereofinclude those having (i) the CDRs of any one of SEQ ID NO:1-6, 12, 13,46-48, or 50-52 in combination with the CDRs of any one of SEQ IDNO:7-11, 14, or 53-58; (ii) first, second, and third heavy chain CDRsselected from SEQ ID NOS:15-23, 42, and 43 in combination with first,second and third light chain CDRs selected from SEQ ID NOS:24-30, 44,and 45; (iii) a humanized forms of (i) or (ii); (iv) a heavy chaincomprising an amino acid sequence comprising at least 85% sequenceidentity to any one of SEQ ID NO:1 or 2 in combination with a lightchain comprising an amino acid sequence comprising at least 85% sequenceidentity to SEQ ID NO:7 or 8; (v) a humanized form or (iv); or (vi) aheavy chain comprising an amino acid sequence comprising at least 85%sequence identity to any one of SEQ ID NO:3-6, 46-48, or 50-52 incombination with a light chain comprising an amino acid sequencecomprising at least 85% sequence identity to SEQ ID NO:9-11 or 53-58.

In some embodiments, the antibodies and fragments and fusion proteinthereof is CDR1 heavy chain variant having the amino acid residuecorresponding with D31 or N31 of a 3E10 heavy chain amino acid sequenceor a CDR thereof substituted with arginine (R) or lysine (L).

In some embodiments, the antibodies and fragments and fusion proteinthereof include the nucleic acid binding pocket of SEQ ID NOS:92 or 93,or a variant thereof with same or improved ability to bind to a nucleicacid, such as DNA, RNA, or a combination thereof.

Also provided are binding proteins themselves including a CDR1 heavychain variant having the amino acid residue corresponding with D31 orN31 of a 3E10 heavy chain amino acid sequence or the CDR1 thereofsubstituted with arginine (R) or lysine (L), as well as binding proteinsthemselves having the nucleic acid binding pocket of SEQ ID NOS:92 or93, or a variant thereof with same or improved ability to bind to anucleic acid, such as DNA, RNA, or a combination thereof.

In some embodiments, the antibody or fragment or fusion protein can bebispecific, and can, for example, include a binding sequence thattargets a cell type, tissue, or organ of interest.

The nucleic acid cargo can be composed of DNA, RNA, modified nucleicacids, including but not limited to, PNA, or a combination thereof. Thenucleic acid cargo is typically a functional cargo, such as a functionalnucleic (e.g., an inhibitory RNA), an mRNA, or a vector, for example anexpression vector. The nucleic acid cargo, including vectors, caninclude a nucleic acid sequence encoding a polypeptide of interestoperably linked to expression control sequence. The vector can be, forexample, a plasmid. Typically the cargo is not, for example, randomlysheared or fragment genomic DNA.

In some embodiments, the cargo includes or consists of a nucleic acidencoding a Cas endonuclease, a gRNA, or a combination thereof. In someembodiments, the cargo includes or consists of a nucleic acid encoding achimeric antigen receptor polypeptide. In some embodiments, the cargo isa functional nucleic acid such as antisense molecules, siRNA, microRNA(miRNA), aptamers, ribozymes, RNAi, or external guide sequences, or anucleic acid construct encoding the same.

The cargo can include or consist of a plurality of a single nucleic acidmolecule, or a plurality of 2, 3, 4, 5, 6, 7, 8, 9, 10, or moredifferent nucleic acid molecules. In some embodiments, the nucleic acidmolecules of cargo include or consists of nucleic acid molecules betweenabout 1 and about 25,000 nucleobases in length. The cargo can be singlestranded nucleic acids, double stranded nucleic acids, or a combinationthereof.

Pharmaceutical compositions including the complexes and apharmaceutically acceptable excipient are also provided. In someembodiments, the complexes are encapsulated in polymeric nanoparticles.A targeting moiety, a cell-penetrating peptide, or a combination thereofcan be associated with, linked, conjugated, or otherwise attacheddirectly or indirectly to the nanoparticle.

Methods of delivering into cells, the nucleic acid cargo, by contactingthe cells with an effective amount of the complexes alone orencapsulated in nanoparticles are also provided. The contacting canoccur in vitro, ex vivo, or in vivo. In some embodiments, an effectiveamount of ex vivo treated cells are administered to a subject in needthereof, e.g., in an effective amount to treat one or more symptoms of adisease or disorder.

In some embodiments, the contacting occurs in vivo followingadministration to a subject in need thereof. The subject can have adisease or disorder, such as a genetic disorder or cancer. Thecompositions can be administered to the subject, for example byinjection or infusion, in an effective amount to reduce one or moresymptoms of the disease or disorder in the subject.

Applications of the compositions and methods are also provided, andinclude, but are not limited to, gene therapy and CAR T cellmanufacture/formation/therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are scatter plots showing control (1A), and uptake of PNAwhen alone (1B) and when mixed with 3E10 for 1 hour (1C). FIG. 1D is abar graph quantifying the data in FIGS. 1A-1C.

FIGS. 2A-2C are scatter plots showing control (2A), and uptake of PNAwhen alone (2B) and when mixed with 3E10 for 24 hour (2C). FIG. 2D is abar graph quantifying the data in FIGS. 2A-2C.

FIGS. 3A-3C are scatter plots showing control (3A), and uptake of siRNAwhen alone (3B) and when mixed with 3E10 for 24 hour (3C). FIG. 3D is abar graph quantifying the data in FIGS. 3A-3C.

FIGS. 4A-4H are scatter plots showing control (4A), and uptake of mRNAwhen alone (4B) and when mixed at various concentrations with 3E10 for24 hour (4C-4H). FIG. 4I is a bar graph quantifying the data in FIGS.4A-4H.

FIGS. 5A-5H are scatter plots showing control (5A), and uptake of mRNAwhen alone (5B) and when mixed at various concentrations with 3E10 for 1hour (5C-5H). FIG. 5I is a bar graph quantifying the data in FIGS.5A-5H.

FIG. 6 is a series of images showing cellular expression of a GFPreporter plasmid DNA 72 hours after mixture with 3E10 and 24 hours ofincubation with cells.

FIG. 7A is a bar graph showing accumulation in tumors of fluorescentlylabeled siRNA mixed with increasing doses of 3E10 (0.25, 0.5, and 1 mg)for 15 minutes at room temperature prior to systemic injection in mice.FIG. 7B is a bar graph showing accumulation in tumors of fluorescentlylabeled siRNA mixed with 1 mg 3E10 or 0.1 mg D31N variant 3E10 for 15minutes at room temperature prior to systemic injection in mice. Alltumors were analyzed 24 hours after injection.

FIG. 8 is a line graph showing 3E10-mediated delivery of mRNA(bioluminescene (Photons/second)) to mouse muscles (IM) over time (dayspost-IM injection).

FIGS. 9A and 9B are images showing control (FIG. 9A) and distribution ofIV Injected 3E10-D31N to muscle (FIG. 9B), imaged by IVIS (Perkin Elmer)24 hours after injection. FIG. 9C is a bar graph quantifying thefluorescence in the IVIS images.

FIG. 10 is a bar graph quantifying the fluorescence in the IVIS imagesof dose-dependent biodistribution of 3E10-D31N to tissues 24 hoursfollowing 100 μg or 200 μg intravenous injection of 3E10-D31N labeledwith VivoTag680 into mice (Perkin Elmer).

FIGS. 11A and 11B are images showing control (FIG. 11A) and distributionof IV Injected 3E10-D31N to syngeneic colon tumors (CT26) (FIG. 11B),imaged by IVIS (Perkin Elmer) 24 hours after injection. FIG. 11C is abar graph quantifying the fluorescence in the IVIS images.

FIGS. 12A, 12B, and 12C are images showing control (FIG. 12A), anddistribution of IV Injected naked single stranded DNA (ssDNA) (FIG. 12B)and 3E10-D31N+ssDNA (FIG. 12C) syngeneic colon tumors (CT26), imaged byIVIS (Perkin Elmer) 24 hours after injection. FIG. 12D is a bar graphquantifying the fluorescence in the IVIS images.

FIG. 13 is a bar graph showing 3E10-mediated delivery and stimulation ofRIG-I.

FIG. 14A is an illustration of molecular modeling of 3E10, a putativeNucleic Acid Binding pocket (NAB1) thereof, and the predicted structuralchanges induced by amino acid mutations therein. FIG. 14B is anillustration of molecular modeling of 3E10-scFv (Pymol) with NAB1 aminoacid residues highlighted by punctate dots.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

As used herein, the term “single chain Fv” or “scFv” as used hereinmeans a single chain variable fragment that includes a light chainvariable region (VL) and a heavy chain variable region (VH) in a singlepolypeptide chain joined by a linker which enables the scFv to form thedesired structure for antigen binding (i.e., for the VH and VL of thesingle polypeptide chain to associate with one another to form a Fv).The VL and VH regions may be derived from the parent antibody or may bechemically or recombinantly synthesized.

As used herein, the term “variable region” is intended to distinguishsuch domain of the immunoglobulin from domains that are broadly sharedby antibodies (such as an antibody Fc domain). The variable regionincludes a “hypervariable region” whose residues are responsible forantigen binding. The hypervariable region includes amino acid residuesfrom a “Complementarity Determining Region” or “CDR” (i.e., typically atapproximately residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in thelight chain variable domain and at approximately residues 27-35 (H1),50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991))and/or those residues from a “hypervariable loop” (i.e., residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917).

As used herein, the term “Framework Region” or “FR” residues are thosevariable domain residues other than the hypervariable region residues asherein defined.

As used herein, the term “antibody” refers to natural or syntheticantibodies that bind a target antigen. The term includes polyclonal andmonoclonal antibodies. In addition to intact immunoglobulin molecules,also included in the term “antibodies” are binding proteins, fragments,and polymers of those immunoglobulin molecules, and human or humanizedversions of immunoglobulin molecules that bind the target antigen.

As used herein, the term “cell-penetrating antibody” refers to animmunoglobulin protein, fragment, variant thereof, or fusion proteinbased thereon that is transported into the cytoplasm and/or nucleus ofliving mammalian cells. The “cell-penetrating anti-DNA antibody”specifically binds DNA (e.g., single-stranded and/or double-strandedDNA). In some embodiments, the antibody is transported into thecytoplasm of the cells without the aid of a carrier or conjugate. Inother embodiments, the antibody is conjugated to a cell-penetratingmoiety, such as a cell penetrating peptide. In some embodiments, thecell-penetrating antibody is transported in the nucleus with or withouta carrier or conjugate.

In addition to intact immunoglobulin molecules, also included in theterm “antibodies” are fragments, binding proteins, and polymers ofimmunoglobulin molecules, chimeric antibodies containing sequences frommore than one species, class, or subclass of immunoglobulin, such ashuman or humanized antibodies, and recombinant proteins containing aleast the idiotype of an immunoglobulin that specifically binds DNA. Theantibodies can be tested for their desired activity using the in vitroassays described herein, or by analogous methods, after which their invivo therapeutic activities are tested according to known clinicaltesting methods.

As used herein, the term “variant” refers to a polypeptide orpolynucleotide that differs from a reference polypeptide orpolynucleotide, but retains essential properties. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more modifications (e.g.,substitutions, additions, and/or deletions). A substituted or insertedamino acid residue may or may not be one encoded by the genetic code. Avariant of a polypeptide may be naturally occurring such as an allelicvariant, or it may be a variant that is not known to occur naturally.

Modifications and changes can be made in the structure of thepolypeptides of in disclosure and still obtain a molecule having similarcharacteristics as the polypeptide (e.g., a conservative amino acidsubstitution). For example, certain amino acids can be substituted forother amino acids in a sequence without appreciable loss of activity.Because it is the interactive capacity and nature of a polypeptide thatdefines that polypeptide's biological functional activity, certain aminoacid sequence substitutions can be made in a polypeptide sequence andnevertheless obtain a polypeptide with like properties.

In making such changes, the hydropathic index of amino acids can beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a polypeptide is generallyunderstood in the art. It is known that certain amino acids can besubstituted for other amino acids having a similar hydropathic index orscore and still result in a polypeptide with similar biologicalactivity. Each amino acid has been assigned a hydropathic index on thebasis of its hydrophobicity and charge characteristics. Those indicesare: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine(+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8);glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9);tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5);glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9);and arginine (−4.5).

It is believed that the relative hydropathic character of the amino aciddetermines the secondary structure of the resultant polypeptide, whichin turn defines the interaction of the polypeptide with other molecules,such as enzymes, substrates, receptors, antibodies, antigens, andcofactors. It is known in the art that an amino acid can be substitutedby another amino acid having a similar hydropathic index and stillobtain a functionally equivalent polypeptide. In such changes, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those within ±1 are particularly preferred, and those within±0.5 are even more particularly preferred.

Substitution of like amino acids can also be made on the basis ofhydrophilicity, particularly where the biological functional equivalentpolypeptide or peptide thereby created is intended for use inimmunological embodiments. The following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamnine (+0.2); glycine (0); proline (−0.5±1); threonine(−0.4); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood thatan amino acid can be substituted for another having a similarhydrophilicity value and still obtain a biologically equivalent, and inparticular, an immunologically equivalent polypeptide. In such changes,the substitution of amino acids whose hydrophilicity values are within±2 is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include (original residue: exemplary substitution): (Ala:Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln:Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu:Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip:Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). Embodiments of thisdisclosure thus contemplate functional or biological equivalents of apolypeptide as set forth above. In particular, embodiments of thepolypeptides can include variants having about 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99%, or more sequence identity to the polypeptide ofinterest.

As used herein, the term “percent (%) sequence identity” is defined asthe percentage of nucleotides or amino acids in a candidate sequencethat are identical with the nucleotides or amino acids in a referencenucleic acid sequence, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity.Alignment for purposes of determining percent sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriateparameters for measuring alignment, including any algorithms needed toachieve maximal alignment over the full-length of the sequences beingcompared can be determined by known methods.

For purposes herein, the % sequence identity of a given nucleotides oramino acids sequence C to, with, or against a given nucleic acidsequence D (which can alternatively be phrased as a given sequence Cthat has or includes a certain % sequence identity to, with, or againsta given sequence D) is calculated as follows:

100 times the fraction W/Z,

where W is the number of nucleotides or amino acids scored as identicalmatches by the sequence alignment program in that program's alignment ofC and D, and where Z is the total number of nucleotides or amino acidsin D. It will be appreciated that where the length of sequence C is notequal to the length of sequence D, the % sequence identity of C to Dwill not equal the % sequence identity of D to C.

As used herein, the term “specifically binds” refers to the binding ofan antibody to its cognate antigen (for example, DNA) while notsignificantly binding to other antigens. Specific binding of an antibodyto a target under such conditions requires the antibody be selected forits specificity to the target. A variety of immunoassay formats may beused to select antibodies specifically immunoreactive with a particularprotein. For example, solid-phase ELISA immunoassays are routinely usedto select monoclonal antibodies specifically immunoreactive with aprotein. See, e.g., Harlow and Lane (1988) Antibodies, A LaboratoryManual, Cold Spring Harbor Publications, New York, for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity. Preferably, an antibody “specifically binds”to an antigen with an affinity constant (Ka) greater than about 10⁵mol⁻¹ (e.g., 10⁶ mol⁻¹, 10⁷ mol⁻¹, 10⁸ mol⁻¹, 10⁹ mol⁻¹, 10¹⁰ mol⁻¹,10¹¹ mol⁻¹, and 10¹² mol⁻¹ or more) with that second molecule.

As used herein, the term “monoclonal antibody” or “MAb” refers to anantibody obtained from a substantially homogeneous population ofantibodies, i.e., the individual antibodies within the population areidentical except for possible naturally occurring mutations that may bepresent in a small subset of the antibody molecules.

As used herein, the term “subject” means any individual who is thetarget of administration. The subject can be a vertebrate, for example,a mammal. Thus, the subject can be a human. The term does not denote aparticular age or sex.

As used herein, the term “effective amount” means that the amount of thecomposition used is of sufficient quantity to ameliorate one or morecauses or symptoms of a disease or disorder. Such amelioration onlyrequires a reduction or alteration, not necessarily elimination. Theprecise dosage will vary according to a variety of factors such assubject-dependent variables (e.g., age, immune system health, etc.), thedisease or disorder being treated, as well as the route ofadministration and the pharmacokinetics of the agent being administered.

As used herein, the term “pharmaceutically acceptable” refers to amaterial that is not biologically or otherwise undesirable, i.e., thematerial may be administered to a subject without causing anyundesirable biological effects or interacting in a deleterious mannerwith any of the other components of the pharmaceutical composition inwhich it is contained.

As used herein, the term “carrier” or “excipient” refers to an organicor inorganic ingredient, natural or synthetic inactive ingredient in aformulation, with which one or more active ingredients are combined. Thecarrier or excipient would naturally be selected to minimize anydegradation of the active ingredient and to minimize any adverse sideeffects in the subject, as would be well known to one of skill in theart.

As used herein, the term “treat” refers to the medical management of apatient with the intent to cure, ameliorate, stabilize, or prevent adisease, pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder.

As used herein, “targeting moiety” is a substance which can direct aparticle or molecule to a receptor site on a selected cell or tissuetype, can serve as an attachment molecule, or serve to couple or attachanother molecule. As used herein, “direct” refers to causing a moleculeto preferentially attach to a selected cell or tissue type. This can beused to direct cellular materials, molecules, or drugs, as discussedbelow.

As used herein, the term “inhibit” or “reduce” means to decrease anactivity, response, condition, disease, or other biological parameter.This can include, but is not limited to, the complete ablation of theactivity, response, condition, or disease. This may also include, forexample, a 10% reduction in the activity, response, condition, ordisease as compared to the native or control level. Thus, the reductioncan be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount ofreduction in between as compared to native or control levels.

As used herein, a “fusion protein” refers to a polypeptide formed by thejoining of two or more polypeptides through a peptide bond formedbetween the amino terminus of one polypeptide and the carboxyl terminusof another polypeptide. The fusion protein can be formed by the chemicalcoupling of the constituent polypeptides or it can be expressed as asingle polypeptide from a nucleic acid sequence encoding the singlecontiguous fusion protein. A single chain fusion protein is a fusionprotein having a single contiguous polypeptide backbone. Fusion proteinscan be prepared using conventional techniques in molecular biology tojoin the two genes in frame into a single nucleic acid sequence, andthen expressing the nucleic acid in an appropriate host cell underconditions in which the fusion protein is produced.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein.

Use of the term “about” is intended to describe values either above orbelow the stated value in a range of approx. +/−10%; in otherembodiments the values may range in value either above or below thestated value in a range of approx. +/−5%; in other embodiments thevalues may range in value either above or below the stated value in arange of approx. +/−2%; in other embodiments the values may range invalue either above or below the stated value in a range of approx.+/−1%. The preceding ranges are intended to be made clear by context,and no further limitation is implied.

All methods described herein can be performed in any suitable orderunless otherwise indicated or otherwise clearly contradicted by context.The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the embodimentsand does not pose a limitation on the scope of the embodiments unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

II. Compositions

It has been discovered that 3E10 antibody helps deliver nucleic acidsacross the plasma membrane and into cell cytoplasm and nuclei. Thus,compositions and methods for using 3E10 to enhance delivery of nucleicacid constructs are provided. Typically an effective amount of 3E10antibody is contacted with a nucleic acid whose delivery into cells isdesired. Typically, the contacting occurs for a sufficient amount totime for the 3E10 and the nucleic acid cargo to form a complex. Thecomplexes are contacted with cells for a sufficient amount of time forthe nucleic acid cargo to be delivered into the cells. The cargo mayaccumulate in a greater quantity, greater quality (e.g., more intact,functional, etc.), or a faster rate, or combination thereof, than if thecells were contacted with the nucleic acid cargo in the absence of theantibody. Because the antibody serves as the delivery means, thedelivery systems are typically non-viral.

A. 3E10 Antibodies

Although generally referred to herein as “3E10” or “3E10 antibodies,” itwill be appreciated that fragments and binding proteins, includingantigen-binding fragments, variants, and fusion proteins such as scFv,di-scFv, tr-scFv, and other single chain variable fragments, and othercell-penetrating, nucleic acid transporting molecules disclosed hereinare encompassed by the phrase are also expressly provided for use incompositions and methods disclosed herein. Thus, the antibodies andother binding proteins are also referred to herein as cell-penetrating.

In preferred embodiments, the 3E10 antibody is transported into thecytoplasm and/or nucleus of the cells without the aid of a carrier orconjugate. For example, the monoclonal antibody 3E10 and activefragments thereof that are transported in vivo to the nucleus ofmammalian cells without cytotoxic effect are disclosed in U.S. Pat. Nos.4,812,397 and 7,189,396 to Richard Weisbart.

In some embodiments, the antibody may bind and/or inhibit Rad51. See forexample, the antibody described in Turchick, et al., Nucleic Acids Res.,45(20): 11782-11799 (2017), WO 2020/047344, and WO 2020/047353, each ofwhich is specifically incorporated by reference herein, in its entirety.

Antibodies that can be used in the compositions and methods includewhole immunoglobulin (i.e., an intact antibody) of any class, fragmentsthereof, and synthetic proteins containing at least the antigen bindingvariable domain of an antibody. The variable domains differ in sequenceamong antibodies and are used in the binding and specificity of eachparticular antibody for its particular antigen. However, the variabilityis not usually evenly distributed through the variable domains ofantibodies. It is typically concentrated in three segments calledcomplementarity determining regions (CDRs) or hypervariable regions bothin the light chain and the heavy chain variable domains. The more highlyconserved portions of the variable domains are called the framework(FR). The variable domains of native heavy and light chains eachcomprise four FR regions, largely adopting a beta-sheet configuration,connected by three CDRs, which form loops connecting, and in some casesforming part of, the beta-sheet structure. The CDRs in each chain areheld together in close proximity by the FR regions and, with the CDRsfrom the other chain, contribute to the formation of the antigen bindingsite of antibodies. Therefore, the antibodies typically contain at leastthe CDRs necessary to maintain DNA binding and/or interfere with DNArepair.

The 3E10 antibody is typically a monoclonal 3E10, or a variant,derivative, fragment, fusion, or humanized form thereof that binds thesame or different epitope(s) as 3E10.

A deposit according to the terms of the Budapest Treaty of a hybridomacell line producing monoclonal antibody 3E10 was received on Sep. 6,2000, and accepted by, American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110-2209, USA, and given PatentDeposit Number PTA-2439.

Thus, the antibody may have the same or different epitope specificity asmonoclonal antibody 3E10 produced by ATCC No. PTA 2439 hybridoma. Theantibody can have the paratope of monoclonal antibody 3E10. The antibodycan be a single chain variable fragment of 3E10, or a variant, e.g., aconservative variant thereof. For example, the antibody can be a singlechain variable fragment of 3E10 (3E10 Fv), or a variant thereof.

1. 3E10 Sequences

Amino acid sequences of monoclonal antibody 3E10 are known in the art.For example, sequences of the 3E10 heavy and light chains are providedbelow, where single underlining indicates the CDR regions identifiedaccording to the Kabat system, and in SEQ ID NOS:12-14 italics indicatesthe variable regions and double underlining indicates the signalpeptide. CDRs according to the IMGT system are also provided.

a. 3E10 Heavy Chain

In some embodiments, a heavy chain variable region of 3E10 is:

EVQLVESGGGLVKPGGSRKLSCAASGFTFS DYGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARRGLLLDYWGQGTTLTVSS (SEQ ID NO: 1; Zack, et al.,Immunology and Cell Biology, 72:513-520 (1994);GenBank: L16981.1 - Mouse Ig rearranged L-chaingene, partial cds; and GenBank: AAA65679.1 -immunoglobulin heavy chain, partial  [ Mus musculus ]).

In some embodiments, a 3E10 heavy chain is expressed as

MGWSCIILFLVATATGVHS EVQLVESGGGLVKPGGSRKLSCAASGFTFS

YGMH WVRQAPEKGLEWVA YISSGSSTIYYADTVKG RFTISRDNAKNTLFL QMTSLRSEDTAMYYCARRGLLLDY WGQGTTLTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (3E10 WT Heavy Chain; SEQ ID NO: 12).

Variants of the 3E10 antibody which incorporate mutations into the wildtype sequence are also known in the art, as disclosed for example, inZack, et al., J. Immunol., 157(5):2082-8 (1996). For example, amino acidposition 31 of the heavy chain variable region of 3E10 has beendetermined to be influential in the ability of the antibody andfragments thereof to penetrate nuclei and bind to DNA (bolded in SEQ IDNOS:1, 2 and 13). A D31N mutation (bolded in SEQ ID NOS:2 and 13) inCDR1 penetrates nuclei and binds DNA with much greater efficiency thanthe original antibody (Zack, et al., Immunology and Cell Biology,72:513-520 (1994), Weisbart, et al., J. Autoimmun., 11, 539-546 (1998);Weisbart, Int. J. Oncol., 25, 1867-1873 (2004)). In some embodiments,the antibody has the D31N substitution.

In some embodiments, an amino acid sequence for a preferred variant of aheavy chain variable region of 3E10 is:

(SEQ ID NO: 2) EVQLVESGGGLVKPGGSRKLSCAASGFTFS NYGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARRG LLLDYWGQGTTLTVSS.

In some embodiments, a 3E10 heavy chain is expressed as

MGWSCIILFLVATATGVHS EVQLVESGGGLVKPGGSRKLSCAASGFTFS

YGMH WVRQAPEKGLEWVA YISSGSSTIYYADTVKG RFTISRDNAKNTL FLQMTSLRSEDTAMYYCARRGLLLDY WGQGTTLTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (3E10 D31N Variant Heavy Chain; SEQ ID NO: 13).

In some embodiments, the C-terminal serine of SEQ ID NOS:1 or 2 isabsent or substituted, with, for example, an alanine, in 3E10 heavychain variable region.

The complementarity determining regions (CDRs) as identified by Kabatare shown with underlining above and include CDR H1.1 (originalsequence): DYGMH (SEQ ID NO:15); CDR H1.2 (with D31N mutation): NYGMH(SEQ ID NO:16); CDR H2.1: YISSGSSTIYYADTVKG (SEQ ID NO:17); CDR H3.1:RGLLLDY (SEQ ID NO:18).

Variants of Kabat CDR H2.1 include YISSGSSTIYYADSVKG (SEQ ID NO:19) andYISSSSSTIYYADSVKG (SEQ ID NO:42).

Additionally, or alternatively, the heavy chain complementaritydetermining regions (CDRs) can be defined according to the IMGT system.The complementarity determining regions (CDRs) as identified by the IMGTsystem include CDR H1.3 (original sequence): GFTFSDYG (SEQ ID NO:20);CDR H1.4 (with D31N mutation): GFTFSNYG (SEQ ID NO:21); CDR H2.2:ISSGSSTI (SEQ ID NO:22) and variant ISSSSSTI (SEQ ID NO:43); CDR H3.2:ARRGLLLDY (SEQ ID NO:23).

b. 3E10 Light Chain

In some embodiments, a light chain variable region of 3F10 is:

(SEQ ID NO: 7) DIVLTQSPASLAVSLGQRATISCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSREF PWTFGGGTKLEIK.

An amino acid sequence for the light chain variable region of 3E10 canalso be:

(SEQ ID NO: 8) DIVLTQSPASLAVSLGQRATISCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPARFSGSGSGTDFHLNIHPVEEEDAATYYCQHSREF PWTFGGGTKLELK.

In some embodiments, a 3E10 light chain is expressed as

MGWSCIILFLVATATGVHS DIVLTQSPASLAVSLGQRATISC RASKSVS TSSYSYMHWYQQKPGQPPKLLIK YASYLES GVPARFSGSGSGTDFTLN IHPVEEEDAATYYC QHSREFPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(3E10 WT Light Chain; SEQ ID NO: 14)

Other 3E10 light chain sequences are known in the art. See, for example,Zack, et al., J. Immunol., 15; 154(4):1987-94 (1995); GenBank:L16981.1—Mouse Ig rearranged L-chain gene, partial cds; GenBank:AAA65681.1—immunoglobulin light chain, partial [Mus musculus]).

The complementarity determining regions (CDRs) as identified by Kabatare shown with underlining, including CDR L1.1: RASKSVSTSSYSYMH (SEQ IDNO:24); CDR L2.1: YASYLES (SEQ ID NO:25); CDR L3.1: QHSREFPWT (SEQ IDNO:26).

Variants of Kabat CDR L1.1 include RASKSVSTSSYSYLA (SEQ ID NO:27) andRASKTVSTSSYSYMH (SEQ ID NO:44).

A variant of Kabat CDR L2.1 is YASYLQS (SEQ ID NO:28).

Additionally, or alternatively, the heavy chain complementaritydetermining regions (CDRs) can be defined according to the IMGT system.The complementarity determining regions (CDRs) as identified by the IMGTsystem include CDR L1.2 KSVSTSSYSY (SEQ ID NO:29) and variant KTVSTSSYSY(SEQ ID NO:45); CDR L2.2: YAS (SEQ ID NO:30); CDR L3.2: QHSREFPWT (SEQID NO:26).

In some embodiments, the C-terminal end of sequence of SEQ ID NOS:7 or 8further includes an arginine in the 3E10 light chain variable region.

2. Humanized 3E10

In some embodiments, the antibody is a humanized antibody. Methods forhumanizing non-human antibodies are well known in the art. Generally, ahumanized antibody has one or more amino acid residues introduced intoit from a source that is non-human. These non-human amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Antibody humanization techniquesgenerally involve the use of recombinant DNA technology to manipulatethe DNA sequence encoding one or more polypeptide chains of an antibodymolecule.

Exemplary 3E10 humanized sequences are discussed in WO 2015/106290, WO2016/033324, WO 2019/018426, and WO/2019/018428, and provided below.

a. Humanized 3E10 Heavy Chain Variable Regions

In some embodiments, a humanized 3E10 heavy chain variable domainincludes

(hVH1, SEQ ID NO: 3) EVQLVQSGGGLIQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSYISSGSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR RGLLLDYWGQGTTVTVSS, or(hVH2, SEQ ID NO: 4) EVQLVESGGGLIQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSYISSGSSTIYYADSVKGRFTISRDNSKNTLYLQMTSLRAEDTAVYYCAR RGLLLDYWGQGTTLTVSS, or(hVH3, SEQ ID NO: 5) EVQLQESGGGVVQPGGSLRLSCAASGFTFSNYGMHWIRQAPGKGLEWVSYISSGSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCAR RGLLLDYWGQGTLVTVSS, or(hVH4, SEQ ID NO: 6) EVQLVESGGGLVQPGGSLRLSCSASGFTFSNYGMHWVRQAPGKGLEYVSYISSGSSTIYYADTVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCVK RGLLLDYWGQGTLVTVSS, or(variants 2, 6 and 10, SEQ ID NO: 46)EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR RGLLLDYWGQGTTVTVSS, or(variants 3, 7 and 11, SEQ ID NO: 47)EVQLVESGGGVVQPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSSSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR RGLLLDYWGQGTTVTVSS, or(variants 4, 8 and 12, SEQ ID NO: 48)EVQLVESGGGDVKPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSSSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR RGLLLDYWGQGTTVTVSS, or(variants 13, 16 and 19, SEQ ID NO: 50)EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSYISSGSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR RGLLLDYWGQGTTVTVSS, or(variants 14 and 17, SEQ ID NO: 51)EVQLVESGGGVVQPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSGSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR RGLLLDYWGQGTTVTVSS, or(variants 15 and 18, SEQ ID NO: 52)EVQLVESGGGDVKPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSGSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR RGLLLDYWGQGTTVTVSS.

b. Humanized 3E10 Light Chain Variable Regions

In some embodiments, a humanized 3E10 light chain variable domainincludes

(hVL1, SEQ ID NO: 9) DIQMTQSPSSLSASVGDRVTITCRASKSVSTSSYSYLAWYQQKPEKAPKLLIKYASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREF PWTFGAGTKLELK, or(hVL2, SEQ ID NO: 10) DIQMTQSPSSLSASVGDRVTISCRASKSVSTSSYSYMHWYQQKPEKAPKLLIKYASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHSREF PWTFGAGTKLELK, or(hVL3, SEQ ID NO: 11) DIVLTQSPASLAVSPGQRATITCRASKSVSTSSYSYMHWYQQKPGQPPKLLIYYASYLESGVPARFSGSGSGTDFTLTINPVEANDTANYYCQHSREF PWTFGQGTKVEIK(variants 2, 3 and 4, SEQ ID NO: 53)DIQMTQSPSSLSASLGDRATITCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQHSREF PWTFGGGTKVEIK(variants 6, 7 and 8, SEQ ID NO: 54)DIQMTQSPSSLSASLGDRATITCRASKSVSTSSYSYMHWYQQKPGQAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREF PWTFGQGTKVEIK(variants 10, 11 and 12, SEQ ID NO: 55)DIQMTQSPSSLSASVGDRVTITCRASKSVSTSSYSYMHWYQQKPGKAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREF PWTFGQGTKVEIK(variants 13, 14 and 15, SEQ ID NO: 56)DIQMTQSPSSLSASLGDRATITCRASKTVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQHSREF PWTFGGGTKVEIK(variants 16, 17 and 18, SEQ ID NO: 57)DIQMTQSPSSLSASVGDRVTITCRASKTVSTSSYSYMHWYQQKPGKAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREF PWTFGQGTKVEIK(variant 19, SEQ ID NO: 58)DIQMTQSPSSLSASLGDRATITCRASKTVSTSSYSYMHWYQQKPGQAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREF PWTFGQGTKVEIK

c. Cell Penetration and Nuclear Localization

The disclosed compositions and methods typically utilize antibodies thatmaintain the ability to penetrate cells, and optionally nuclei.

The mechanisms of cellular internalization by autoantibodies arediverse. Some are taken into cells through electrostatic interactions orFcR-mediated endocytosis, while others utilize mechanisms based onassociation with cell surface myosin or calreticulin, followed byendocytosis (Ying-Chyi et al., Eur J Immunol 38, 3178-3190 (2008),Yanase et al., J Clin Invest 100, 25-31 (1997)). 3E10 penetrates cellsin an Fc-independent mechanism (as evidenced by the ability of 3E10fragments lacking an Fc to penetrate cells) but involves presence of thenucleoside transporter ENT2 (Weisbart et al., Sci Rep 5:12022. doi:10.1038/srep12022. (2015), Zack et al., J Immunol 157, 2082-2088 (1996),Hansen et al., J Biol Chem 282, 20790-20793 (2007)). Thus, in someembodiments, the antibodies utilized in the disclosed compositions andmethods are ones that penetrates cells in an Fc-independent mechanismbut involves presence of the nucleoside transporter ENT2.

Mutations in 3E10 that interfere with its ability to bind DNA may renderthe antibody incapable of nuclear penetration. Thus, typically thedisclosed variants and humanized forms of the antibody maintain theability to bind nucleic acids, particularly DNA. In addition, 3E10 scFvhas previously been shown capable of penetrating into living cells andnucleic in an ENT2-dependent manner, with efficiency of uptake impairedin ENT2-deficient cells (Hansen, et al., J. Biol. Chem. 282, 20790-20793(2007)). Thus, in some embodiments, the disclosed variants and humanizedforms of the antibody maintain the ability penetrate into cell nuclei inan ENT-dependent, preferably ENT2-dependent manner.

As discussed in WO 2019/152806 and WO 2019/152808 some humanized 3E10variant were found to penetrate cell nuclei more efficiently than theoriginal murine 3E10 (D31N) di-scFv, while others were found to havelost the ability to penetrate nuclei. In particular, variants 10 and 13penetrated nuclei very well compared to the murine antibody.

Potential bipartite nuclear localization signals (NLS) in humanized 3E10VL have been identified and may include part or all of the followingsequences:

(SEQ ID NO: 88) RASK S VSTSSYSYMHWYQQKPGQPPKLLIKY; (SEQ ID NO: 89) RASKT VSTSSYSYMHWYQQKPGQPPKLLIKY; or (SEQ ID NO: 90)RVTITCRASKSVSTSSYSYMHWYQQKPGKAPKL.

An exemplary consensus NLS can be, or include,(X)RASKTVSTSSYSYMHWYQQKPGQPPKLL(X)KY (where (X)=any residue, butpreferentially is a basic residue (R or K) (SEQ ID NO:91) or a variantthereof with at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99percent sequence identity to SEQ ID NO:53.

Thus, in some embodiments, particularly where nuclear importation isimportant, the disclosed antibodies may include the sequence of any oneof SEQ ID NOS:88-91, or fragments and variants thereof (e.g., 70, 75,80, 85, 90, 95, 96, 97, 98, 99, or 100% amino acid sequence identitywith any one of SEQ ID NOS:88-91) that can translocate into the nucleusof a cell.

Presence of an NLS indicates that 3E10 may cross the nuclear envelopevia the nuclear import pathway. In some embodiments, the NLS improvesimportation by interacting with one or more members of the importpathway. Thus, in some embodiments, the NLS can bind to importin-β, animportin-β/importin-α heterodimer, or a combination thereof.

3. Nucleic Acid Binding

The disclosed compositions and methods typically utilize antibodies thatmaintain the ability to bind nucleic acids such as DNA, RNA, or acombination thereof.

The Examples below illustrate molecular modeling of 3E10 and additional3E10 variants. Molecular modeling of 3E10 (Pymol) revealed a putativeNucleic Acid Binding pocket (NAB1) (see, e.g., FIGS. 14A and 14B), andillustrated with underlining the sequences below.

WT HEAVY CHAIN scFv SEQUENCE (SEQ ID NO: 92)E VQLVESGGGL VKPGGSRKLS CAASGFTFSD YGMHWVRQAPEKGLEWVAYI SSGSSTIYYA DTVKGRFTIS RDNAKNTLFLQMTSLRSEDT AMYYCARRGL LLDYWGQGTT LTVS LIGHT CHAIN scFv SEQUENCE(SEQ ID NO: 93) D IVLTQSPASL AVSLGQRATI SCRASKSVST SSYSYMHWYQQKPGQPPKLL IKYASYLESG VPARFSGSGS GTDFTLNIHPVEEEDAATYY CQHSREFPWT FGGGTKLEIK RADAAPGGGG SGGGGSGGGGS

In some embodiments, the disclosed antibodies include some or all of theunderlined NAB1 sequences. In some embodiments, the antibodies include avariant sequence that has an altered ability of bind nucleic acids. Insome embodiments, the mutations (e.g., substitutions, insertions, and/ordeletions) in the NAB1 improve binding of the antibody to nucleic acidssuch as DNA, RNA, or a combination thereof. In some embodiments, themutations are conservative substitutions. In some embodiments, themutations increase the cationic charge of the NAB1 pocket.

As discussed and exemplified herein, mutation of aspartic acid atresidue 31 of CDR1 to asparagine increased the cationic charge of thisresidue and enhanced nucleic acid binding and delivery in vivo(3E10-D31N).

Additional exemplary variants include mutation of aspartic acid atresidue 31 of CDR1 to arginine (3E10-D31R), which modeling indicatesexpands cationic charge, or lysine (3E10-D31K) which modeling indicateschanges charge orientation. Thus, in some embodiments, the 3E10 bindingprotein includes a D31R or D31K substitution.

All of the sequences disclosed herein having the residue correspondingto 3E10 D31 or N31, are expressly disclosed with a D31R or D31K or N31Ror N31K substitution therein.

Molecular modeling of 3E10 (Pymol) revealed a putative Nucleic AcidBinding pocket (NAB1) (FIGS. 14A-14B). Mutation of aspartic acid atresidue 31 of CDR1 to asparagine increased the cationic charge of thisresidue and enhanced nucleic acid binding and delivery in vivo(3E10-D31N).

Mutation of aspartic acid at residue 31 of CDR1 to arginine (3E10-D31R),further expanded the cationic charge while mutation to lysine(3E10-D31K) changed charge orientation (FIG. 14A).

NAB1 amino acids predicted from molecular modeling have been underlinedin the heavy and light chain sequences above. FIG. 14B is anillustration showing molecular modeling of 3E10-scFv (Pymol) with NAB1amino acid residues illustrated with punctate dots.

4. Fragments, Variants, and Fusion Proteins

The anti-DNA antibody can be composed of an antibody fragment or fusionprotein including an amino acid sequence of a variable heavy chainand/or variable light chain that is at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to the amino acid sequence of the variable heavy chain and/orlight chain of 3E10 or a humanized form thereof (e.g., any of SEQ IDNOS:1-11 or 46-58, or the heavy and/or light chains of any of SEQ IDNOS:12-14).

The anti-DNA antibody can be composed of an antibody fragment or fusionprotein that includes one or more CDR(s) that is at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100% identical to the amino acid sequence of the CDR(s) of 3E10,or a variant or humanized form thereof (e.g., CDR(s) of any of SEQ IDNOS:1-11 or 46-58, or SEQ ID NOS:12-14, or SEQ ID NOS:15-30 or 42-45).The determination of percent identity of two amino acid sequences can bedetermined by BLAST protein comparison. In some embodiments, theantibody includes one, two, three, four, five, or all six of the CDRs ofthe above-described preferred variable domains.

Preferably, the antibody includes one of each of a heavy chain CDR1,CDR2, and CDR3 in combination with one of each of a light chain CDR1,CDR2, and CDR3.

Predicted complementarity determining regions (CDRs) of the light chainvariable sequence for 3E10 are provided above. See also GenBank:AAA65681.1—immunoglobulin light chain, partial [Mus musculus] andGenBank: L34051.1—Mouse Ig rearranged kappa-chain mRNA V-region.Predicted complementarity determining regions (CDRs) of the heavy chainvariable sequence for 3E10 are provide above. See also, for example,Zack, et al., Immunology and Cell Biology, 72:513-520 (1994), GenBankAccession number AAA65679.1. Zach, et al., J. Immunol. 154 (4),1987-1994 (1995) and GenBank: L16982.1—Mouse Ig rearranged H-chain gene,partial cds.

Thus, in some embodiments, the cell-penetrating antibody contains theCDRs, or the entire heavy and light chain variable regions, of SEQ IDNO:1 or 2, or the heavy chain region of SEQ ID NO:12 or 13; or ahumanized form thereof in combination with SEQ ID NO:7 or 8, or thelight chain region of SEQ ID NO:14; or a humanized form thereof. In someembodiments, the cell-penetrating antibody contains the CDRs, or theentire heavy and light chain variable regions, of SEQ ID NO:3, 4, 5, or6 in combination with SEQ ID NO:9, 10, or 11. In some embodiments, thecell-penetrating antibody contains the CDRs, or the entire heavy andlight chain variable regions, of any one of SEQ ID NO:46-48 or 50-52 incombination with any one of SEQ ID NO:53-58.

All of the sequences disclosed herein having the residue correspondingto 3E10 D31 or N31, are expressly disclosed with a D31R or D31K or N31Ror N31K substitution therein. Thus, in some embodiments, the 3E10binding protein is a variant of any of the foregoing or followingsequences wherein the amino acid residue corresponding to residue 31 ofthe 3E10 heavy chain is substituted with arginine (R) or lysine (K).

Also included are fragments of antibodies which have bioactivity. Thefragments, whether attached to other sequences or not, includeinsertions, deletions, substitutions, or other selected modifications ofparticular regions or specific amino acids residues, provided theactivity of the fragment is not significantly altered or impairedcompared to the nonmodified antibody or antibody fragment.

Techniques can also be adapted for the production of single-chainantibodies specific to an antigenic protein of the present disclosure.Methods for the production of single-chain antibodies are well known tothose of skill in the art. A single chain antibody can be created byfusing together the variable domains of the heavy and light chains usinga short peptide linker, thereby reconstituting an antigen binding siteon a single molecule. Single-chain antibody variable fragments (scFvs)in which the C-terminus of one variable domain is tethered to theN-terminus of the other variable domain via a 15 to 25 amino acidpeptide or linker have been developed without significantly disruptingantigen binding or specificity of the binding. The linker is chosen topermit the heavy chain and light chain to bind together in their properconformational orientation.

The anti-DNA antibodies can be modified to improve their therapeuticpotential. For example, in some embodiments, the cell-penetratinganti-DNA antibody is conjugated to another antibody specific for asecond therapeutic target in the cytoplasm and/or nucleus of a targetcell. For example, the cell-penetrating anti-DNA antibody can be afusion protein containing 3E10 Fv and a single chain variable fragmentof a monoclonal antibody that specifically binds the second therapeutictarget. In other embodiments, the cell-penetrating anti-DNA antibody isa bispecific antibody having a first heavy chain and a first light chainfrom 3E10 and a second heavy chain and a second light chain from amonoclonal antibody that specifically binds the second therapeutictarget.

Bispecific antibodies and other binding proteins having a first heavychain and a first light chain from 3E10 and a second heavy chain and asecond light chain from a monoclonal antibody that specifically binds asecond target are discussed in Weisbart, et al., Mol. Cancer Ther.,11(10):2169-73 (2012), and Weisbart, et al., Int. J. Oncology, 25:1113-8(2004), and U.S. Patent Application No. 2013/0266570, which arespecifically incorporated by reference in their entireties. In someembodiments, the second target is specific for a target cell-type,tissue, organ etc. Thus the second heavy chain and second light chaincan serve as a targeting moiety that targets the complex to the targetcell-type, tissue, organ. In some embodiments, the second heavy chainand second light chain target, hematopoietic stem cells, CD34⁺ cells, Tcells or any another preferred cell type, e.g., by targeting a receptoror ligand expressed on the preferred cell type. In some embodiments, thesecond heavy chain and second light chain target the thymus, spleen, orcancer cells.

In some embodiments, particularly those for targeting T cell in vivo,for example, for in vivo production of CAR T cells, immune cell or Tcell markers such as CD3, CD7, or CD8 can be targeted. For example,anti-CD8 antibodies and anti-CD3 Fab fragments have both been used totarget T cells in vivo (Pfeiffer, et al., EMBO Mol Med., 10(11) (2018).pii: e9158. doi: 10.15252/emmm 201809158., Smith, et al., NatNanotechnol., 12(8):813-820 (2017). doi: 10.1038/nnano 2017.57). Thus,in some embodiments, the 3E10 antibody or antigen binding fragment orfusion protein is a bispecific antibody part of which can bindspecifically to CD3, CD7, CD8, or another immune cell (e.g., T cell)marker, or a marker for a specific tissue such as the thymus, spleen, orliver.

Divalent single-chain variable fragments (di-scFvs) can be engineered bylinking two scFvs. This can be done by producing a single peptide chainwith two VH and two VL regions, yielding tandem scFvs. ScFvs can also bedesigned with linker peptides that are too short for the two variableregions to fold together (about five amino acids), forcing scFvs todimerize. This type is known as diabodies. Diabodies have been shown tohave dissociation constants up to 40-fold lower than correspondingscFvs, meaning that they have a much higher affinity to their target.Still shorter linkers (one or two amino acids) lead to the formation oftrimers (triabodies or tribodies). Tetrabodies have also been produced.They exhibit an even higher affinity to their targets than diabodies. Insome embodiments, the anti-DNA antibody may contain two or more linkedsingle chain variable fragments of 3E10 (e.g., 3E10 di-scFv, 3E10tri-scFv), or conservative variants thereof. In some embodiments, theanti-DNA antibody is a diabody or triabody (e.g., 3E10 diabody, 3E10triabody). Sequences for single and two or more linked single chainvariable fragments of 3E10 are provided in WO 2017/218825 and WO2016/033321.

The function of the antibody may be enhanced by coupling the antibody ora fragment thereof with a therapeutic agent. Such coupling of theantibody or fragment with the therapeutic agent can be achieved bymaking an immunoconjugate or by making a fusion protein, or by linkingthe antibody or fragment to a nucleic acid such as DNA or RNA (e.g.,siRNA), comprising the antibody or antibody fragment and the therapeuticagent.

A recombinant fusion protein is a protein created through geneticengineering of a fusion gene. This typically involves removing the stopcodon from a cDNA sequence coding for the first protein, then appendingthe cDNA sequence of the second protein in frame through ligation oroverlap extension PCR. The DNA sequence will then be expressed by a cellas a single protein. The protein can be engineered to include the fullsequence of both original proteins, or only a portion of either. If thetwo entities are proteins, often linker (or “spacer”) peptides are alsoadded which make it more likely that the proteins fold independently andbehave as expected.

In some embodiments, the cell-penetrating antibody is modified to alterits half-life. In some embodiments, it is desirable to increase thehalf-life of the antibody so that it is present in the circulation or atthe site of treatment for longer periods of time. For example, it may bedesirable to maintain titers of the antibody in the circulation or inthe location to be treated for extended periods of time. In otherembodiments, the half-life of the anti-DNA antibody is decreased toreduce potential side effects. Antibody fragments, such as 3E10Fv mayhave a shorter half-life than full size antibodies. Other methods ofaltering half-life are known and can be used in the described methods.For example, antibodies can be engineered with Fc variants that extendhalf-life, e.g., using Xtend™ antibody half-life prolongation technology(Xencor, Monrovia, Calif.).

a. Linkers

The term “linker” as used herein includes, without limitation, peptidelinkers. The peptide linker can be any size provided it does notinterfere with the binding of the epitope by the variable regions. Insome embodiments, the linker includes one or more glycine and/or serineamino acid residues. Monovalent single-chain antibody variable fragments(scFvs) in which the C-terminus of one variable domain are typicallytethered to the N-terminus of the other variable domain via a 15 to 25amino acid peptide or linker. The linker is chosen to permit the heavychain and light chain to bind together in their proper conformationalorientation. Linkers in diabodies, triabodies, etc., typically include ashorter linker than that of a monovalent scFv as discussed above. Di-,tri-, and other multivalent scFvs typically include three or morelinkers. The linkers can be the same, or different, in length and/oramino acid composition. Therefore, the number of linkers, composition ofthe linker(s), and length of the linker(s) can be determined based onthe desired valency of the scFv as is known in the art. The linker(s)can allow for or drive formation of a di-, tri-, and other multivalentscFv.

For example, a linker can include 4-8 amino acids. In a particularembodiment, a linker includes the amino acid sequence GQSSRSS (SEQ IDNO:31). In another embodiment, a linker includes 15-20 amino acids, forexample, 18 amino acids. In a particular embodiment, the linker includesthe amino acid sequence GQSSRSSSGGGSSGGGGS (SEQ ID NO:32). Otherflexible linkers include, but are not limited to, the amino acidsequences Gly-Ser, Gly-Ser-Gly-Ser (SEQ ID NO:33), Ala-Ser,Gly-Gly-Gly-Ser (SEQ ID NO:34), (Gly₄-Ser)₂ (SEQ ID NO:35) and(Gly₄-Ser)₄ (SEQ ID NO:36), and (Gly-Gly-Gly-Gly-Ser)₃ (SEQ ID NO:37).

Other exemplary linkers include, for example, RADAAPGGGGSGGGGSGGGGS (SEQID NO:59) and ASTKGPSVFPLAPLESSGS (SEQ ID NO:60).

b. Exemplary Anti-DNA scFv Sequences

Exemplary murine 3E10 scFv sequences, including mono-, di-, and tri-scFvare disclosed in WO 2016/033321, WO 2017/218825, WO 2019/018426, andWO/2019/018428, and provided below. Cell-penetrating antibodies for usein the disclosed compositions and methods include exemplary scFv, andfragments and variants thereof.

The amino acid sequence for scFv 3E10 (D31N) is:

(SEQ ID NO: 38) AGIHDIVLTQSPASLAVSLGQRATISCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSREFPWTFGGGTKLEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSRKLSCAASGFTFSNYGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARRGLLLDYWGQGTTLTVSSLEQ KLISEEDLNSAVDHHHHHH.

Annotation of scFv Protein Domains with Reference to SEQ ID NO:38

-   -   AGIH sequence increases solubility (amino acids 1-4 of SEQ ID        NO:38)    -   Vk variable region (amino acids 5-115 of SEQ ID NO:38)    -   Initial (6 aa) of light chain CH1 (amino acids 116-121 of SEQ ID        NO:38)    -   (GGGGS)₃ (SEQ ID NO:37) linker (amino acids 122-136 of SEQ ID        NO:38)    -   VH variable region (amino acids 137-252 of SEQ ID NO:38)    -   Myc tag (amino acids 253-268 SEQ ID NO:38)    -   His 6 tag (amino acids 269-274 of SEQ ID NO:38)

Amino Acid Sequence of 3E10 Di-scFv (D31N)

Di-scFv 3E10 (D31N) is a di-single chain variable fragment including 2Xthe heavy chain and light chain variable regions of 3E10 and wherein theaspartic acid at position 31 of the heavy chain is mutated to anasparagine. The amino acid sequence for di-scFv 3E10 (D31N) is:

(SEQ ID NO: 39) AGIHDIVLTQSPASLAVSLGQRATISCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSREFPWTFGGGTKLEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSRKLSCAASGFTFSNYGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARRGLLLDYWGQGTTLTVSSASTKGPSVFPLAPLESSGSDIVLTQSPASLAVSLGQRATISCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSREFPWTFGGGTKLEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSRKLSCAASGFTFSNYGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARRGLLLDYWGQGTTLTVSSLEQKLISEEDLNSAVDHHHHHH.

Annotation of Di-scFv Protein Domains with Reference to SEQ ID NO:39

-   -   AGIH sequence increases solubility (amino acids 1-4 of SEQ ID        NO:39)    -   Vk variable region (amino acids 5-115 of SEQ ID NO:39)    -   Initial (6 aa) of light chain CH1 (amino acids 116-121 of SEQ ID        NO:39)    -   (GGGGS)₃ (SEQ ID NO:37) linker (amino acids 122-136 of SEQ ID        NO:39)    -   VH variable region (amino acids 137-252 of SEQ ID NO:39)    -   Linker between Fv fragments consisting of human IgG CH1 initial        13 amino acids (amino acids 253-265 of SEQ ID NO:39)    -   Swivel sequence (amino acids 266-271 of SEQ ID NO:39)    -   Vk variable region (amino acids 272-382 of SEQ ID NO:39)    -   Initial (6 aa) of light chain CH1 (amino acids 383-388 of SEQ ID        NO:39)    -   (GGGGS)₃ (SEQ ID NO:37) linker (amino acids 389-403 of SEQ ID        NO:39)    -   VH variable region (amino acids 404-519 of SEQ ID NO:39)    -   Myc tag (amino acids 520-535 of SEQ ID NO:39)    -   His 6 tag (amino acids 536-541 of SEQ ID NO:39)

Amino Acid Sequence for Tri-scFv

Tri-scFv 3E10 (D31N) is a tri-single chain variable fragment including3X the heavy chain and light chain variable regions of 310E and whereinthe aspartic acid at position 31 of the heavy chain is mutated to anasparagine. The amino acid sequence for tri-scFv 3E10 (D31N) is:

(SEQ ID NO: 40) AGIHDIVLTQSPASLAVSLGQRATISCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSREFPWTFGGGTKLEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSRKLSCAASGFTFSNYGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARRGLLLDYWGQGTTLTVSSASTKGPSVFPLAPLESSGSDIVLTQSPASLAVSLGQRATISCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSREFPWTFGGGTKLEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSRKLSCAASGFTFSNYGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARRGLLLDYWGQGTTLTVSSASTKGPSVFPLAPLESSGSDIVLTQSPASLAVSLGQRATISCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSREFPWTFGGGTKLEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSRKLSCAASGFTFSNYGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARRGLLLDYWGQGTTLTVSSLEQKLISEEDLNSAVDHHHHHH.

Annotation of Tri-scFv Protein Domains with Reference to SEQ ID NO:40

-   -   AGIH sequence increases solubility (amino acids 1˜4 of SEQ ID        NO:40)    -   Vk variable region (amino acids 5-115 of SEQ ID NO:40)    -   Initial (6 aa) of light chain CH1 (amino acids 116-121 of SEQ ID        NO:40)    -   (GGGGS)₃ (SEQ ID NO:37) linker (amino acids 122-136 of SEQ ID        NO:40)    -   VH variable region (amino acids 137-252 of SEQ ID NO:40)    -   Linker between Fv fragments consisting of human IgG CH1 initial        13 amino acids (amino acids 253-265 of SEQ ID NO:40)    -   Swivel sequence (amino acids 266-271 of SEQ ID NO:40)    -   Vk variable region (amino acids 272-382 of SEQ ID NO:40)    -   Initial (6 aa) of light chain CH1 (amino acids 383-388 of SEQ ID        NO:40)    -   (GGGGS)₃ (SEQ ID NO:37) linker (amino acids 389-403 of SEQ ID        NO:40)    -   VH variable region (amino acids 404-519 of SEQ ID NO:40)    -   Linker between Fv fragments consisting of human IgG C_(H)1        initial 13 amino acids (amino acids 520-532 of SEQ ID NO:40)    -   Swivel sequence (amino acids 533-538 of SEQ ID NO:40)    -   Vk variable region (amino acids 539-649 of SEQ ID NO:40)    -   Initial (6 aa) of light chain CH1 (amino acids 650-655 of SEQ ID        NO:40)    -   (GGGGS)₃ (SEQ ID NO:37) linker (amino acids 656-670 of SEQ ID        NO:40)    -   VH variable region (amino acids 671-786 of SEQ ID NO:40)    -   Myc tag (amino acids 787-802 of SEQ ID NO:40)    -   His 6 tag (amino acids 803-808 of SEQ ID NO:40)

WO 2016/033321 and Noble, et al., Cancer Research, 75(11):2285-2291(2015), show that di-scFv and tri-scFv have some improved and additionalactivities compared to their monovalent counterpart. The subsequencescorresponding to the different domains of each of the exemplary fusionproteins are also provided above. One of skill in the art willappreciate that the exemplary fusion proteins, or domains thereof, canbe utilized to construct fusion proteins discussed in more detail above.For example, in some embodiments, the di-scFv includes a first scFvincluding a Vk variable region (e.g., amino acids 5-115 of SEQ ID NO:39,or a functional variant or fragment thereof), linked to a VH variabledomain (e.g., amino acids 137-252 of SEQ ID NO:39, or a functionalvariant or fragment thereof), linked to a second scFv including a Vkvariable region (e.g., amino acids 272-382 of SEQ ID NO:39, or afunctional variant or fragment thereof), linked to a VH variable domain(e.g., amino acids 404-519 of SEQ ID NO:39, or a functional variant orfragment thereof). In some embodiments, a tri-scFv includes a di-scFvlinked to a third scFv domain including a Vk variable region (e.g.,amino acids 539-649 of SEQ ID NO:40, or a functional variant or fragmentthereof), linked to a VH variable domain (e.g., amino acids 671-786 ofSEQ ID NO:40, or a functional variant or fragment thereof).

The Vk variable regions can be linked to VH variable domains by, forexample, a linker (e.g., (GGGGS)₃ (SEQ ID NO:37), alone or incombination with a (6 aa) of light chain CH1 (amino acids 116-121 of SEQID NO:39). Other suitable linkers are discussed above and known in theart. scFv can be linked by a linker (e.g., human IgG CH1 initial 13amino acids (253-265) of SEQ ID NO:39), alone or in combination with aswivel sequence (e.g., amino acids 266-271 of SEQ ID NO:39). Othersuitable linkers are discussed above and known in the art.

Therefore, a di-scFv can include amino acids 5-519 of SEQ ID NO:39. Atri-scFv can include amino acids 5-786 of SEQ ID NO:40. In someembodiments, the fusion proteins include additional domains. Forexample, in some embodiments, the fusion proteins include sequences thatenhance solubility (e.g., amino acids 1-4 of SEQ ID NO:39). Therefore,in some embodiments, a di-scFv can include amino acids 1-519 of SEQ IDNO:39. A tri-scFv can include amino acids 1-786 of SEQ ID NO:40. In someembodiments that fusion proteins include one or more domains thatenhance purification, isolation, capture, identification, separation,etc., of the fusion protein. Exemplary domains include, for example, Myctag (e.g., amino acids 520-535 of SEQ ID NO:39) and/or a His tag (e.g.,amino acids 536-541 of SEQ ID NO:39). Therefore, in some embodiments, adi-scFv can include the amino acid sequence of SEQ ID NO:39. A tri-scFvcan include the amino acid sequence of SEQ ID NO:40. Other substitutabledomains and additional domains are discussed in more detail above.

An exemplary 3E10 humanized Fv sequence is discussed in WO 2016/033324:

(SEQ ID NO: 41) DIVLTQSPASLAVSPGQRATITCRASKSVSTSSYSYMHWYQQKPGQPPKLLIYYASYLESGVPARFSGSGSGTDFTLTINPVEANDTANYYCQHSREFPWTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCSASGFTFSNYGMHWVRQAPGKGLEYVSYISSGSSTIYYADTVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCVKRGLLLDYWGQGTLVTVSS.

Exemplary 3E10 humanized di-scFv sequences are discussed in WO2019/018426 and WO/2019/018428, and include:

(Variant 2, SEQ ID NO: 61)DIQMTQSPSSLSASLGDRATITCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQHSREFPWTFGGGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTTVTVSSASTKGPSVFPLAPLESSGSDIQMTQSPSSLSASLGDRATITCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQHSREFPWTFGGGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTT VTVSS,(Variant 3, SEQ ID NO: 62)DIQMTQSPSSLSASLGDRATITCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQHSREFPWTFGGGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSSSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTTVTVSSASTKGPSVFPLAPLESSGSDIQMTQSPSSLSASLGDRATITCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQHSREFPWTFGGGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSSSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTT VTVSS,(Variant 4, SEQ ID NO: 63)DIQMTQSPSSLSASLGDRATITCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQHSREFPWTFGGGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGDVKPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSSSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTTVTVSSASTKGPSVFPLAPLESSGSDIQMTQSPSSLSASLGDRATITCRASKSVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQHSREFPWTFGGGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGDVKPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSSSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTT VTVSS,(Variant 6, SEQ ID NO: 64)DIQMTQSPSSLSASLGDRATITCRASKSVSTSSYSYMHWYQQKPGQAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREFPWTFGQGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTTVTVSSASTKGPSVFPLAPLESSGSDIQMTQSPSSLSASLGDRATITCRASKSVSTSSYSYMHWYQQKPGQAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREFPWTFGQGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTT VTVSS,(Variant 7, SEQ ID NO: 65)DIQMTQSPSSLSASLGDRATITCRASKSVSTSSYSYMHWYQQKPGQAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREFPWTFGQGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSSSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTTVTVSSASTKGPSVFPLAPLESSGSDIQMTQSPSSLSASLGDRATITCRASKSVSTSSYSYMHWYQQKPGQAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREFPWTFGQGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSSSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTT VTVSS,(Variant 8, SEQ ID NO: 66)DIQMTQSPSSLSASLGDRATITCRASKSVSTSSYSYMHWYQQKPGQAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREFPWTFGQGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGDVKPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSSSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTTVTVSSASTKGPSVFPLAPLESSGSDIQMTQSPSSLSASLGDRATITCRASKSVSTSSYSYMHWYQQKPGQAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREFPWTFGQGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGDVKPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSSSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTT VTVSS,(Variant 10, SEQ ID NO: 67)DIQMTQSPSSLSASVGDRVTITCRASKSVSTSSYSYMHWYQQKPGKAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREFPWTFGQGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTTVTVSSASTKGPSVFPLAPLESSGSDIQMTQSPSSLSASVGDRVTITCRASKSVSTSSYSYMHWYQQKPGKAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREFPWTFGQGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTT VTVSS,(Variant 11, SEQ ID NO: 68)DIQMTQSPSSLSASVGDRVTITCRASKSVSTSSYSYMHWYQQKPGKAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREFPWTFGQGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSSSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTTVTVSSASTKGPSVFPLAPLESSGSDIQMTQSPSSLSASVGDRVTITCRASKSVSTSSYSYMHWYQQKPGKAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREFPWTFGQGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSSSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTT VTVSS,(Variant 12, SEQ ID NO: 69)DIQMTQSPSSLSASVGDRVTITCRASKSVSTSSYSYMHWYQQKPGKAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREFPWTFGQGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGDVKPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSSSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTTVTVSSASTKGPSVFPLAPLESSGSDIQMTQSPSSLSASVGDRVTITCRASKSVSTSSYSYMHWYQQKPGKAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREFPWTFGQGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGDVKPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSSSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTT VTVSS,(Variant 13, SEQ ID NO: 70)DIQMTQSPSSLSASLGDRATITCRASKTVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQHSREFPWTFGGGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSYISSGSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTTVTVSSASTKGPSVFPLAPLESSGSDIQMTQSPSSLSASLGDRATITCRASKTVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQHSREFPWTFGGGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSYISSGSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTT VTVSS,(Variant 14, SEQ ID NO: 71)DIQMTQSPSSLSASLGDRATITCRASKTVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQHSREFPWTFGGGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSGSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTTVTVSSASTKGPSVFPLAPLESSGSDIQMTQSPSSLSASLGDRATITCRASKTVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQHSREFPWTFGGGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSGSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTT VTVSS,(Variant 15, SEQ ID NO: 72)DIQMTQSPSSLSASLGDRATITCRASKTVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQHSREFPWTFGGGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGDVKPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSGSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTTVTVSSASTKGPSVFPLAPLESSGSDIQMTQSPSSLSASLGDRATITCRASKTVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQHSREFPWTFGGGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGDVKPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSGSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTT VTVSS,(Variant 16, SEQ ID NO: 73)DIQMTQSPSSLSASVGDRVTITCRASKTVSTSSYSYMHWYQQKPGKAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREFPWTFGQGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSYISSGSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTTVTVSSASTKGPSVFPLAPLESSGSDIQMTQSPSSLSASVGDRVTITCRASKTVSTSSYSYMHWYQQKPGKAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREFPWTFGQGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSYISSGSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTT VTVSS,(Variant 17, SEQ ID NO: 74)DIQMTQSPSSLSASVGDRVTITCRASKTVSTSSYSYMHWYQQKPGKAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREFPWTFGQGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSGSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTTVTVSSASTKGPSVFPLAPLESSGSDIQMTQSPSSLSASVGDRVTITCRASKTVSTSSYSYMHWYQQKPGKAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREFPWTFGQGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSGSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTT VTVSS,(Variant 18, SEQ ID NO: 75)DIQMTQSPSSLSASVGDRVTITCRASKTVSTSSYSYMHWYQQKPGKAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREFPWTFGQGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGDVKPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSGSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTTVTVSSASTKGPSVFPLAPLESSGSDIQMTQSPSSLSASVGDRVTITCRASKTVSTSSYSYMHWYQQKPGKAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREFPWTFGQGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGDVKPGGSLRLSCAASGFTFSNYGMHWVRQAPEKGLEWVSYISSGSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTT VTVSS, and(Variant 19, SEQ ID NO: 76)DIQMTQSPSSLSASLGDRATITCRASKTVSTSSYSYMHWYQQKPGQAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREFPWTFGQGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSYISSGSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTTVTVSSASTKGPSVFPLAPLESSGSDIQMTQSPSSLSASLGDRATITCRASKTVSTSSYSYMHWYQQKPGQAPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSREFPWTFGQGTKVEIKRADAAPGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSYISSGSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTT VTVSS.

c. Additional Sequences

Additional sequences that may used in the construction of anti-DNAantigen binding proteins, antibodies, fragments and fusion proteinsinclude, but are not limited to,

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSYISSGSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (IgG1L2345A/L235A heavy chain full length sequence, SEQ ID NO: 77),ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV(IgG1 constant heavy region 1, SEQ ID NO: 78),EPKSCDKTHTCP (IgG1 hinge region, SEQ ID NO: 79),PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (IgG1 L2345A/L235A constant heavy region 2, SEQ ID NO: 80),GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 constant heavy region 3, SEQ ID NO: 81),EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSYISSGSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYDSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (IgG1 N297Dheavy chain full length sequence, SEQ ID NO: 82),PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYDSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (IgG1 N297D constant heavy region 2, SEQ ID NO: 83),EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSYISSGSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYDSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (IgG1L2345A/L235A/N297D heavy chain full length sequence, SEQ ID NO: 84),PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYDSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (IgG1 L2345A/L235A/N297D constant heavy region 2,SEQ ID NO: 85), PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 86, Unmodified constant heavy region 2), andDIQMTQSPSSLSASLGDRATITCRASKTVSTSSYSYMHWYQQKPGQPPKLLIKYASYLESGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQHSREFPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Light chain full length sequence, SEQ ID NO: 87).

B. Cargo

As used in the methods provided herein, the 3E10 is typically contactedwith cells in complex with a nucleic acid cargo. The interaction betweenthe antibody or binding protein and the nucleic acid cargo isnon-covalent.

The nucleic acid cargo can be single stranded or double stranded. Thenucleic acid cargo can be or include DNA, RNA, nucleic acid analogs, ora combination thereof. As discussed in more detail below, nucleic acidanalogs can be modified at the base moiety, sugar moiety, or phosphatebackbone. Such modification can improve, for example, stability,hybridization, or solubility of the nucleic acid.

The nucleic acid cargo is typically functional in the sense that is orencodes an agent that is biologically active once delivered into cells.Exemplary cargo is discussed in more detail below, but includes, forexample, mRNA or DNA encoding polypeptides of interest including, forexample expression constructs and vectors, inhibitory nucleic acids suchas siRNA, or nucleic acid encoding the inhibitory nucleic acidincluding, for example expression constructs and vectors.

The disclosed compositions can include a plurality of a single nucleicacid cargo molecule. In some embodiments, the compositions include aplurality of a multiplicity (e.g., 2, 3, 4, 5, 6, 7, 8, 9 10, or more)of different nucleic acid molecules.

In some embodiments, the cargo molecules are 0.001, 0.01, 1, 10's 100's,1,000's, 10,000's, and/or 100,000's of kilobases in length.

In some embodiments, e.g., the cargo may be between 0.001 kb and 100 kb,or between 0.001 kb and 50 kb, or between 0.001 kb and 25 kb, or between0.001 kb and 12.5 kb, or between 0.001 kb and 10 kb, or between 0.001 kband 8 kb, or 0.001 kb and 5 kb, or between 0.001 kb and 2.5 kb, orbetween 0.001 kb and 1 kb, or between 0.01 kb and 100 kb, or between0.01 kb and 50 kb, or between 0.01 kb and 25 kb, or between 0.01 kb and12.5 kb, or between 0.01 kb and 10 kb, or between 0.01 kb and 8 kb, or0.01 kb and 5 kb, or between 0.01 kb and 2.5 kb, or between 0.01 kb and1 kb, or between 0.1 kb and 100 kb, or between 0.1 kb and 50 kb, orbetween 0.1 kb and 25 kb, or between 0.1 kb and 12.5 kb, or between 0.1kb and 10 kb, or between 0.1 kb and 8 kb, or 0.1 kb and 5 kb, or between0.1 kb and 2.5 kb, or between 0.1 kb and 1 kb, or between 1 kb and 100kb, or between 1 kb and 50 kb, or between 1 kb and 25 kb, or between 1kb and 12.5 kb, or between 1 kb and 10 kb, or between 1 kb and 8 kb, or1 kb and 5 kb, or between 1 kb and 2.5 kb, each inclusive.

In some embodiments, e.g., the cargo may be between 0.2 kb and 10 kb, orbetween 0.2 kb and 5 kb, or between 0.2 kb and 2.5 kb, or between 0.2 kband 1 kb, or between 0.2 kb and 0.5 kb, or between 0.2 kb and 0.25 kb,or between 0.5 kb and 10 kb, or between 0.5 kb and 5 kb, or between 1 kband 5 kb, or between 1 kb and 3 kb, or between 2 kb and 10 kb, orbetween 3 kb and 5 kb.

It will be appreciated that for specific application the nucleic acidcargo may be one or more discrete lengths that, for example, fallswithin one of the foregoing ranges (inclusive), the specific values foreach are expressly disclosed. For example, the size can be as small as asingle nucleotide or nucleobase. In an exemplary application the cargois a cyclic dinucleotide like cGAMP, which is a STING agonist. In otherembodiments, the cargo is a short oligomer. For example, oligomers asshort as 8-mers can be used for anti-sense or splice switching. Slightlylonger ones (e.g., 18 to 20 mers) can be used for gene editing.

1. Forms of the Cargo

The nucleic acid cargo is a nucleic acid and can be an isolated nucleicacid composition. As used herein, “isolated nucleic acid” refers to anucleic acid that is separated from other nucleic acid molecules thatare present in a mammalian genome, including nucleic acids that normallyflank one or both sides of the nucleic acid in a mammalian genome. Theterm “isolated” as used herein with respect to nucleic acids alsoincludes the combination with any non-naturally-occurring nucleic acidsequence, since such non-naturally-occurring sequences are not found innature and do not have immediately contiguous sequences in anaturally-occurring genome.

An isolated nucleic acid can be, for example, a DNA molecule, providedone of the nucleic acid sequences normally found immediately flankingthat DNA molecule in a naturally-occurring genome is removed or absent.Thus, an isolated nucleic acid includes, without limitation, a DNAmolecule that exists as a separate molecule independent of othersequences (e.g., a chemically synthesized nucleic acid, or a cDNA orgenomic DNA fragment produced by PCR or restriction endonucleasetreatment), as well as recombinant DNA that is incorporated into avector, an autonomously replicating plasmid, a virus (e.g., aretrovirus, lentivirus, adenovirus, or herpes virus), or into thegenomic DNA of a prokaryote or eukaryote. In addition, an isolatednucleic acid can include an engineered nucleic acid such as arecombinant DNA molecule that is part of a hybrid or fusion nucleicacid. A nucleic acid existing among hundreds to millions of othernucleic acids within, for example, a cDNA library or a genomic library,or a gel slice containing a genomic DNA restriction digest, is not to beconsidered an isolated nucleic acid.

The nucleic acid sequences encoding polypeptides include genomicsequences. Also disclosed are mRNA/cDNA sequence wherein the exons havebeen deleted. Other nucleic acid sequences encoding polypeptides, suchpolypeptides that include the above-identified amino acid sequences andfragments and variants thereof, are also disclosed. Nucleic acidsencoding polypeptides may be optimized for expression in the expressionhost of choice. Codons may be substituted with alternative codonsencoding the same amino acid to account for differences in codon usagebetween the organism from which the nucleic acid sequence is derived andthe expression host. In this manner, the nucleic acids may besynthesized using expression host-preferred codons.

Nucleic acids can be in sense or antisense orientation, or can be, forexample, complementary to a reference sequence encoding a polypeptide.

a. Vectors

The cargo can be a vector, for example, a vector encoding apolypeptide(s) and/or functional nucleic acid(s). Nucleic acids, such asthose described above, can be inserted into vectors for expression incells. As used herein, a “vector” is a replicon, such as a plasmid,phage, virus or cosmid, into which another DNA segment may be insertedso as to bring about the replication of the inserted segment. Vectorscan be expression vectors. An “expression vector” is a vector thatincludes one or more expression control sequences, and an “expressioncontrol sequence” is a DNA sequence that controls and regulates thetranscription and/or translation of another DNA sequence.

Nucleic acids in vectors can be operably linked to one or moreexpression control sequences. For example, the control sequence can beincorporated into a genetic construct so that expression controlsequences effectively control expression of a coding sequence ofinterest. Examples of expression control sequences include promoters,enhancers, and transcription terminating regions. A promoter is anexpression control sequence composed of a region of a DNA molecule,typically within 100 nucleotides upstream of the point at whichtranscription starts (generally near the initiation site for RNApolymerase II). To bring a coding sequence under the control of apromoter, it is necessary to position the translation initiation site ofthe translational reading frame of the polypeptide between one and aboutfifty nucleotides downstream of the promoter. Enhancers provideexpression specificity in terms of time, location, and level. Unlikepromoters, enhancers can function when located at various distances fromthe transcription site. An enhancer also can be located downstream fromthe transcription initiation site. A coding sequence is “operablylinked” and “under the control” of expression control sequences in acell when RNA polymerase is able to transcribe the coding sequence intomRNA, which then can be translated into the protein encoded by thecoding sequence.

Suitable expression vectors include, without limitation, plasmids,cosmids, and viral vectors derived from, for example, bacteriophage,baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalo virus,retroviruses, vaccinia viruses, adenoviruses, and adeno-associatedviruses. Numerous vectors and expression systems are commerciallyavailable from such corporations as Novagen (Madison, Wis.), Clontech(Palo Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen LifeTechnologies (Carlsbad, Calif.).

In some embodiments, the cargo is delivered into the cell and remainsextrachromosomal. In some embodiments, the cargo is introduced into ahost cell and is integrated into the host cell's genome. As discussed inmore detail below, the compositions can be used in methods of genetherapy. Methods of gene therapy can include the introduction into thecell of a polynucleotide that alters the genotype of the cell.Introduction of the polynucleotide can correct, replace, or otherwisealter the endogenous gene via genetic recombination. Methods can includeintroduction of an entire replacement copy of a defective gene, aheterologous gene, or a small nucleic acid molecule such as anoligonucleotide. For example, a corrective gene can be introduced into anon-specific location within the host's genome.

In some embodiments, the cargo is a vector. Methods to constructexpression vectors containing genetic sequences and appropriatetranscriptional and translational control elements are well known in theart. These methods include in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Expressionvectors generally contain regulatory sequences and necessary elementsfor the translation and/or transcription of the inserted codingsequence, which can be, for example, the polynucleotide of interest. Thecoding sequence can be operably linked to a promoter and/or enhancer tohelp control the expression of the desired gene product. Promoters usedin biotechnology are of different types according to the intended typeof control of gene expression. They can be generally divided intoconstitutive promoters, tissue-specific or development-stage-specificpromoters, inducible promoters, and synthetic promoters.

For example, in some embodiments, a polynucleotide of interest isoperably linked to a promoter or other regulatory elements known in theart. Thus, the cargo can be a vector such as an expression vector. Theengineering of polynucleotides for expression in a prokaryotic oreukaryotic system may be performed by techniques generally known tothose of skill in recombinant expression. An expression vector typicallyincludes one of the disclosed compositions under the control of one ormore promoters. To bring a coding sequence “under the control of” apromoter, one positions the 5′ end of the translational initiation siteof the reading frame generally between about 1 and 50 nucleotides“downstream” of (i.e., 3′ of) the chosen promoter. The “upstream”promoter stimulates transcription of the inserted DNA and promotesexpression of the encoded recombinant protein or functional nucleicacid. This is the meaning of “recombinant expression” in the contextused here.

Many standard techniques are available to construct expression vectorscontaining the appropriate nucleic acids andtranscriptional/translational control sequences in order to achieveprotein or peptide or functional nucleic acid expression in a variety ofhost-expression systems.

Expression vectors for use in mammalian cells ordinarily include anorigin of replication (as necessary), a promoter located in front of thegene to be expressed, along with any necessary ribosome binding sites,RNA splice sites, polyadenylation site, and transcriptional terminatorsequences. The origin of replication may be provided either byconstruction of the vector to include an exogenous origin, such as maybe derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV)source, or may be provided by the host cell chromosomal replicationmechanism. If the vector is integrated into the host cell chromosome,the latter is often sufficient.

The promoters may be derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). Further, itis also possible, and may be desirable, to utilize promoter or controlsequences normally associated with the desired gene sequence, providedsuch control sequences are compatible with the host cell systems.

A number of viral based expression systems may be utilized, for example,commonly used promoters are derived from polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40 (SV40). The early and late promotersof SV40 virus are useful because both are obtained easily from the virusas a fragment which also contains the SV40 viral origin of replication.Smaller or larger SV40 fragments may also be used, provided there isincluded the approximately 250 bp sequence extending from the HindIIIsite toward the BgII site located in the viral origin of replication.

In cases where an adenovirus is used as an expression vector, the codingsequences may be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene may then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing proteins in infectedhosts.

Specific initiation signals may also be required for efficienttranslation of the disclosed compositions. These signals include the ATGinitiation codon and adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may additionally need to beprovided. One of ordinary skill in the art would readily be capable ofdetermining this need and providing the necessary signals. It is wellknown that the initiation codon must be in-frame (or in-phase) with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements or transcriptionterminators.

In eukaryotic expression, one will also typically desire to incorporateinto the transcriptional unit an appropriate polyadenylation site if onewas not contained within the original cloned segment. Typically, thepoly A addition site is placed about 30 to 2000 nucleotides “downstream”of the termination site of the protein at a position prior totranscription termination.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably expressconstructs encoding proteins may be engineered. Rather than usingexpression vectors that contain viral origins of replication, host cellscan be transformed with vectors controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched medium, and then areswitched to a selective medium. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into their chromosomes and grow to form foci,which in turn can be cloned and expanded into cell lines.

b. mRNAs

The cargo can be mRNA.

Chemical structures with the ability to promote stability and/ortranslation efficiency may also be used. For example, the RNA can have5′ and 3′ UTRs. The length of the 3′ UTR can, for example, exceed 100nucleotides. In some embodiments the 3′ UTR sequence is between 100 and5000 nucleotides. In some embodiments, the 5′ UTR is between zero and3000 nucleotides in length. The length of 5′ and 3′ UTR sequences to beadded to the coding region can be altered by different methods,including, but not limited to, designing primers for PCR that anneal todifferent regions of the UTRs. Using this approach, one of ordinaryskill in the art can modify the 5′ and 3′ UTR lengths required toachieve optimal translation efficiency following delivery of thetranscribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the gene of interest. Alternatively, UTR sequences that are notendogenous to the gene of interest can be added by incorporating the UTRsequences into the forward and reverse primers or by any othermodifications of the template. The use of UTR sequences that are notendogenous to the gene of interest can be useful for modifying thestability and/or translation efficiency of the RNA. For example, it isknown that AU-rich elements in 3′ UTR sequences can decrease thestability of mRNA. Therefore, 3′ UTRs can be selected or designed toincrease the stability of the transcribed RNA based on properties ofUTRs that are well known in the art.

In some embodiments, the 5′ UTR contains the Kozak sequence of theendogenous gene. Alternatively, when a 5′ UTR that is not endogenous tothe gene of interest is being added by PCR as described above, aconsensus Kozak sequence can be redesigned by adding the 5′ UTRsequence. Kozak sequences can increase the efficiency of translation ofsome RNA transcripts, but does not appear to be required for all RNAs toenable efficient translation. The requirement for Kozak sequences formany mRNAs is known in the art. In other embodiments the 5′ UTR can bederived from an RNA virus whose RNA genome is stable in cells. In otherembodiments various nucleotide analogues can be used in the 3′ or 5′ UTRto impede exonuclease degradation of the mRNA.

In some embodiments, the mRNA has a cap on the 5′ end, a 3′ poly(A)tail, or a combination thereof which determine ribosome binding,initiation of translation and stability mRNA in the cell.

5′caps provide stability to RNA molecules. The 5′ cap may, for example,be m⁷G(5′)ppp(5′)G, m⁷G(5′)ppp(5′)A, G(5′)ppp(5′)G or G(5′)ppp(5′)A capanalogs, which are all commercially available. The 5′ cap can also be ananti-reverse-cap-analog (ARCA) (Stepinski, et al., RNA, 7:1468-95(2001)) or any other suitable analog. The 5′ cap can be incorporatedusing techniques known in the art (Cougot, et al., Trends in Biochem.Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001);Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

The RNAs can also contain an internal ribosome entry site (IRES)sequence. The IRES sequence may be any viral, chromosomal orartificially designed sequence which initiates cap-independent ribosomebinding to mRNA and facilitates the initiation of translation.

Generally, the length of a poly(A) tail positively correlates with thestability of the transcribed RNA. In one embodiment, the poly(A) tail isbetween 100 and 5000 adenosines.

A polyA segment can be produced during PCR by using a reverse primercontaining a polyT tail, such as 100 T tail (size can be, e.g., 50-5000T), or after PCR by any other method, including, but not limited to, DNAligation or in vitro recombination. Poly(A) tails also provide stabilityto RNAs and reduce their degradation. Poly(A) tails of RNAs canadditionally or alternatively be extended following in vitrotranscription with the use of a poly(A) polymerase, such as E. colipolyA polymerase (E-PAP).

Additionally, the attachment of different chemical groups to the 3′ endcan increase mRNA stability. Such attachment can containmodified/artificial nucleotides, aptamers and other compounds. Forexample, ATP analogs can be incorporated into the poly(A) tail usingpoly(A) polymerase. ATP analogs can further increase the stability ofthe RNA. Suitable ATP analogs include, but are not limited to,cordiocipin and 8-azaadenosine.

2. Sequence of the Cargo

a. Polypeptide of Interest

The cargo can encode one or more proteins. The cargo can be apolynucleotide that can be monocistronic or polycistronic. In someembodiments, polynucleotide is multigenic. The polynucleotide can be,for example, an mRNA or a expression construct such as a vector.

The cargo can encode one or more polypeptides of interest. Thepolypeptide can be any polypeptide. For example, the polypeptide encodedby the polynucleotide can be a polypeptide that provides a therapeuticor prophylactic effect to an organism or that can be used to diagnose adisease or disorder in an organism. For example, for treatment ofcancer, autoimmune disorders, parasitic, viral, bacterial, fungal orother infections, the polynucleotide(s) to be expressed may encode apolypeptide that functions as a ligand or receptor for cells of theimmune system, or can function to stimulate or inhibit the immune systemof an organism.

In some embodiments, the polynucleotide supplements or replaces apolynucleotide that is defective in the organism.

In particular embodiments, the polynucleotide encodes dystrophin,utrophin, or a combination thereof. Such compositions may beadministered in an effective amount to treat a subject from a dystrophy,particularly a muscular dystrophy, for example, Duchenne's musculardystrophy.

In another particular embodiment, the polynucleotide encodes antigen,e.g., an antigen that can be utilized in a vaccine formulation andassociated methods. In a particular embodiment, polynucleotide encodes aviral antigen(s), for example, a SARS-CoV-2 antigen(s). Thus,compositions and methods of use thereof for protection against, and thetreatment of, SARS-CoV-2 virus and viral infections and diseaseassociate therewith including COVID19 are provided.

In some embodiments, the polynucleotide includes a selectable marker,for example, a selectable marker that is effective in a eukaryotic cell,such as a drug resistance selection marker. This selectable marker genecan encode a factor necessary for the survival or growth of transformedhost cells grown in a selective culture medium. Typical selection genesencode proteins that confer resistance to antibiotics or other toxins,e.g., ampicillin, neomycin, methotrexate, kanamycin, gentamycin, Zeocin,or tetracycline, complement auxotrophic deficiencies, or supply criticalnutrients withheld from the media.

In some embodiments, the polynucleotide includes a reporter gene.Reporter genes are typically genes that are not present or expressed inthe host cell. The reporter gene typically encodes a protein whichprovides for some phenotypic change or enzymatic property. Examples ofsuch genes are provided in Weising et al. Ann. Rev. Genetics, 22, 421(1988). Preferred reporter genes include glucuronidase (GUS) gene andGFP genes.

b. Functional Nucleic Acids

The cargo can be or encode a functional nucleic acid. Functional nucleicacids are nucleic acid molecules that have a specific function, such asbinding a target molecule or catalyzing a specific reaction. Asdiscussed in more detail below, functional nucleic acid molecules can bedivided into the following non-limiting categories: antisense molecules,siRNA, miRNA, aptamers, ribozymes, RNAi, and external guide sequences,and cyclic dinucleotides. The functional nucleic acid molecules can actas effectors, inhibitors, modulators, and stimulators of a specificactivity possessed by a target molecule, or the functional nucleic acidmolecules can possess a de novo activity independent of any othermolecules.

Functional nucleic acid molecules can interact with any macromolecule,such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functionalnucleic acids can interact with the mRNA or the genomic DNA of a targetpolypeptide or they can interact with the polypeptide itself. Oftenfunctional nucleic acids are designed to interact with other nucleicacids based on sequence homology between the target molecule and thefunctional nucleic acid molecule. In other situations, the specificrecognition between the functional nucleic acid molecule and the targetmolecule is not based on sequence homology between the functionalnucleic acid molecule and the target molecule, but rather is based onthe formation of tertiary structure that allows specific recognition totake place.

Therefore the compositions can include one or more functional nucleicacids designed to reduce expression of a gene, or a gene productthereof. For example, the functional nucleic acid or polypeptide can bedesigned to target and reduce or inhibit expression or translation of anmRNA; or to reduce or inhibit expression, reduce activity, or increasedegradation of a protein. In some embodiments, the composition includesa vector suitable for in vivo expression of the functional nucleic acid.

i. Antisense

The functional nucleic acids can be or encode antisense molecules.Antisense molecules are designed to interact with a target nucleic acidmolecule through either canonical or non-canonical base pairing. Theinteraction of the antisense molecule and the target molecule isdesigned to promote the destruction of the target molecule through, forexample, RNAse H mediated RNA-DNA hybrid degradation. Alternatively theantisense molecule is designed to interrupt a processing function thatnormally would take place on the target molecule, such as transcriptionor replication. Antisense molecules can be designed based on thesequence of the target molecule. There are numerous methods foroptimization of antisense efficiency by finding the most accessibleregions of the target molecule. Exemplary methods include in vitroselection experiments and DNA modification studies using DMS and DEPC.It is preferred that antisense molecules bind the target molecule with adissociation constant (K_(d)) less than or equal to 10⁻⁶, 10⁻⁸, 10⁻¹⁰,or 10⁻¹².

ii. RNA Interference

In some embodiments, the functional nucleic acids induce gene silencingthrough RNA interference. Gene expression can also be effectivelysilenced in a highly specific manner through RNA interference (RNAi).This silencing was originally observed with the addition of doublestranded RNA (dsRNA) (Fire, et al. (1998) Nature, 391:806-11; Napoli, etal. (1990) Plant Cell 2:279-89; Hannon, (2002) Nature, 418:244-51). OncedsRNA enters a cell, it is cleaved by an RNase III—like enzyme, Dicer,into double stranded small interfering RNAs (siRNA) 21-23 nucleotides inlength that contains 2 nucleotide overhangs on the 3′ ends (Elbashir, etal. (2001) Genes Dev., 15:188-200; Bernstein, et al. (2001) Nature,409:363-6; Hammond, et al. (2000) Nature, 404:293-6). In an ATPdependent step, the siRNAs become integrated into a multi-subunitprotein complex, commonly known as the RNAi induced silencing complex(RISC), which guides the siRNAs to the target RNA sequence (Nykanen, etal. (2001) Cell, 107:309-21). At some point the siRNA duplex unwinds,and it appears that the antisense strand remains bound to RISC anddirects degradation of the complementary mRNA sequence by a combinationof endo and exonucleases (Martinez, et al. (2002) Cell, 110:563-74).However, the effect of iRNA or siRNA or their use is not limited to anytype of mechanism.

Short Interfering RNA (siRNA) is a double-stranded RNA that can inducesequence-specific post-transcriptional gene silencing, therebydecreasing or even inhibiting gene expression. In one example, a siRNAtriggers the specific degradation of homologous RNA molecules, such asmRNAs, within the region of sequence identity between both the siRNA andthe target RNA. For example, WO 02/44321 discloses siRNAs capable ofsequence-specific degradation of target mRNAs when base-paired with 3′overhanging ends, herein incorporated by reference for the method ofmaking these siRNAs.

Sequence specific gene silencing can be achieved in mammalian cellsusing synthetic, short double-stranded RNAs that mimic the siRNAsproduced by the enzyme dicer (Elbashir, et al. (2001) Nature, 411:494498) (Ui-Tei, et al. (2000) FEBS Lett 479:79-82). siRNA can bechemically or in vitro-synthesized or can be the result of shortdouble-stranded hairpin-like RNAs (shRNAs) that are processed intosiRNAs inside the cell. Synthetic siRNAs are generally designed usingalgorithms and a conventional DNA/RNA synthesizer. Suppliers includeAmbion (Austin, Tex.), ChemGenes (Ashland, Mass.), Dharmacon (Lafayette,Colo.), Glen Research (Sterling, Va.), MWB Biotech (Esbersberg,Germany), Proligo (Boulder, Colo.), and Qiagen (Vento, The Netherlands).siRNA can also be synthesized in vitro using kits such as Ambion'sSILENCER® siRNA Construction Kit.

The production of siRNA from a vector is more commonly done through thetranscription of a short hairpin RNAse (shRNAs). Kits for the productionof vectors having shRNA are available, such as, for example, Imgenex'sGENESUPPRESSOR™ Construction Kits and Invitrogen's BLOCK-IT™ inducibleRNAi plasmid and lentivirus vectors.

In some embodiment, the functional nucleic acid is siRNA, shRNA, miRNA.In some embodiments, the composition includes a vector expressing thefunctional nucleic acid.

iii. Aptamers

The functional nucleic acids can be or encode an aptamer. Aptamers aremolecules that interact with a target molecule, preferably in a specificway. Typically aptamers are small nucleic acids ranging from 15-50 basesin length that fold into defined secondary and tertiary structures, suchas stem-loops or G-quartets. Aptamers can bind small molecules, such asATP and theophiline, as well as large molecules, such as reversetranscriptase and thrombin. Aptamers can bind very tightly with K_(d)'sfrom the target molecule of less than 10⁻¹² M. It is preferred that theaptamers bind the target molecule with a K_(d) less than 10⁻⁶, 10⁻⁸,10⁻¹⁰, or 10⁻¹². Aptamers can bind the target molecule with a very highdegree of specificity. For example, aptamers have been isolated thathave greater than a 10,000 fold difference in binding affinities betweenthe target molecule and another molecule that differ at only a singleposition on the molecule. It is preferred that the aptamer have a K_(d)with the target molecule at least 10, 100, 1000, 10,000, or 100,000 foldlower than the K_(d) with a background binding molecule. It is preferredwhen doing the comparison for a molecule such as a polypeptide, that thebackground molecule be a different polypeptide.

iv. Ribozymes

The functional nucleic acids can be or encode ribozymes. Ribozymes arenucleic acid molecules that are capable of catalyzing a chemicalreaction, either intramolecularly or intermolecularly. It is preferredthat the ribozymes catalyze intermolecular reactions. There are a numberof different types of ribozymes that catalyze nuclease or nucleic acidpolymerase type reactions which are based on ribozymes found in naturalsystems, such as hammerhead ribozymes. There are also a number ofribozymes that are not found in natural systems, but which have beenengineered to catalyze specific reactions de novo. Preferred ribozymescleave RNA or DNA substrates, and more preferably cleave RNA substrates.Ribozymes typically cleave nucleic acid substrates through recognitionand binding of the target substrate with subsequent cleavage. Thisrecognition is often based mostly on canonical or non-canonical basepair interactions. This property makes ribozymes particularly goodcandidates for target specific cleavage of nucleic acids becauserecognition of the target substrate is based on the target substratessequence.

v. External Guide Sequences

The functional nucleic acids can be or encode external guide sequences.External guide sequences (EGSs) are molecules that bind a target nucleicacid molecule forming a complex, which is recognized by RNase P, whichthen cleaves the target molecule. EGSs can be designed to specificallytarget a RNA molecule of choice. RNAse P aids in processing transfer RNA(tRNA) within a cell. Bacterial RNAse P can be recruited to cleavevirtually any RNA sequence by using an EGS that causes the targetRNA:EGS complex to mimic the natural tRNA substrate. Similarly,eukaryotic EGS/RNAse P-directed cleavage of RNA can be utilized tocleave desired targets within eukaryotic cells. Representative examplesof how to make and use EGS molecules to facilitate cleavage of a varietyof different target molecules are known in the art.

Methods of making and using vectors for in vivo expression of functionalnucleic acids such as antisense oligonucleotides, siRNA, shRNA, miRNA,EGSs, ribozymes, and aptamers are known in the art.

vi. Cyclic Dinucleotides

The functional nucleic acids can be or encode a cyclic dinucleotide.Cyclic dinucleotides bind directly to the STING adaptor protein,resulting in production of IFN-β (Zhang, et al., Mol Cell., 51(2):226-35(2013). doi: 10.1016/j.molce1.2013.05.022.). Several canonical andnoncanonical dinucleotides are known in the art, and include, but arenot limited to, 2′3′-cGAMP, 2′3′-cGAMP, 3′3′-cGAMP, c-di-AMP, c-di-GMP,cAIMP (CL592), cAIMP Difluor (CL614), cAIM(PS)2 Difluor (Rp/Sp) (CL656),2′2′-cGAMP, 2′3′-cGAM(PS)2 (Rp/Sp), 3′3′-cGAMP Fluorinated, c-di-AMPFluorinated, 2′3′-c-di-AMP, 2′3′-c-di-AM(PS)2 (Rp,Rp), 2′3′-c-di-AM(PS)2(Rp,Rp), c-di-GMP Fluorinated, 2′3′-c-di-GMP, c-di-IMP, DMXAA.

vii. Immunostimulatory Oligonucleotides

In some embodiments, the functional nucleic acids can be or encode anoligonucleotide ligand. Examples include, but are not limited to,pattern recognition receptors (PRRs) ligands.

Examples of PRRs include the Toll-like family of signaling moleculesthat play a role in the initiation of innate immune responses and alsoinfluence the later and more antigen specific adaptive immune responses.Therefore, the oligonucleotide can serve as a ligand for a Toll-likefamily signaling molecule, such as Toll-Like Receptor 9 (TLR9).

For example, unmethylated CpG sites can be detected by TLR9 onplasmacytoid dendritic cells and B cells in humans (Zaida, et al.,Infection and Immunity, 76(5):2123-2129, (2008)). Therefore, thesequence of oligonucleotide can include one or more unmethylatedcytosine-guanine (CG or CpG, used interchangeably) dinucleotide motifs.The ‘p’ refers to the phosphodiester backbone of DNA, however, in someembodiments, oligonucleotides including CG can have a modified backbone,for example a phosphorothioate (PS) backbone.

In some embodiments, an oligonucleotide can contain more than one CGdinucleotide, arranged either contiguously or separated by interveningnucleotide(s). The CpG motif(s) can be in the interior of theoligonucleotide sequence. Numerous nucleotide sequences stimulate TLR9with variations in the number and location of CG dinucleotide(s), aswell as the precise base sequences flanking the CG dimers.

Typically, CG ODNs are classified based on their sequence, secondarystructures, and effect on human peripheral blood mononuclear cells(PBMCs). The five classes are Class A (Type D), Class B (Type K), ClassC, Class P, and Class S (Vollmer, J & Krieg, A M, Advanced drug deliveryreviews 61(3): 195-204 (2009), incorporated herein by reference). CGODNs can stimulate the production of Type I interferons (e.g., IFNα) andinduce the maturation of dendritic cells (DCs). Some classes of ODNs arealso strong activators of natural killer (NK) cells through indirectcytokine signaling. Some classes are strong stimulators of human B celland monocyte maturation (Weiner, G L, PNAS USA 94(20): 10833-7 (1997);Dalpke, A H, Immunology 106(1): 102-12 (2002); Hartmann, G, J of Immun.164(3):1617-2 (2000), each of which is incorporated herein byreference).

Other PRR Toll-like receptors include TLR3, and TLR7 which may recognizedouble-stranded RNA, single-stranded and short double-stranded RNAs,respectively, and retinoic acid-inducible gene I (RIG-I)-like receptors,namely RIG-I and melanoma differentiation-associated gene 5 (MDAS),which are best known as RNA-sensing receptors in the cytosol.

RIG-I (retinoic-acid-inducible protein 1, also known as Ddx58) and MDA-5(melanoma-differentiation-associated gene 5, also known as Ifih1 orHelicard) are cytoplasmic RNA helicases that belong to the RIG-I-likereceptors (RLRs) family and are critical for host antiviral responses.

RIG-I and MDA-5 sense double-stranded RNA (dsRNA), a replicationintermediate for RNA viruses, and signal through the mitochondrialantiviral signaling protein MAVS (also known as IPS-1, VISA or Cardif),leading to production of type-I interferons (IFN-α and IFN-β).

RIG-I detects viral RNA that exhibit an uncapped 5′-di/triphosphate endand a short blunt-ended double stranded portion, two essential featuresfacilitating discrimination from self-RNAs. The features of MDA-5physiological ligands have not been fully characterized yet. However, itis admitted that RIG-I and MDA-5 exhibit a different dependency for thelength of dsRNAs: RIG-I selectively binds short dsRNA while MDA-5selectively binds long dsRNA. Consistent with this, RIG-I and MDA-5 bindPoly(I:C), a synthetic dsRNA analog, with different length predilection.

Under some circumstances, RIG-I can also sense dsDNA indirectly. ViraldsDNA can be transcribed by the RNA polymerase III into dsRNA with a5′-triphosphate moiety. Poly(dA:dT), a synthetic analog of B-form DNA,thus constitutes another RIG-I ligand.

Exemplary RIG-I ligands include, but are not limited to, 5′ppp-dsRNA, aspecific agonist of RIG-I; 3p-hpRNA, a specific agonist of RIG-I;Poly(I:C)/LyoVec complexes that are recognized by RIG-I and/or MDA-5depending of the size of poly(I:C); Poly(dA:dT)/LyoVec complexes thatare indirectly recognized by RIG-I.

In some embodiments, the oligonucleotide contains a functional ligandfor TLR3, TLR7, TLR8, TLR9, or RIG-I-like receptors, or combinationsthereof.

Examples of immunostimulatory oligonucleotides, and methods of makingthem are known in the art and commercially available, see for example,Bodera, P. Recent Pat Inflamm Allergy Drug Discov. 5(1):87-93 (2011),incorporated herein by reference.

3. Composition of the Cargo

The disclosed nucleic acid cargo can be or include DNA or RNAnucleotides which typically include a heterocyclic base (nucleic acidbase), a sugar moiety attached to the heterocyclic base, and a phosphatemoiety which esterifies a hydroxyl function of the sugar moiety. Theprincipal naturally-occurring nucleotides include uracil, thymine,cytosine, adenine and guanine as the heterocyclic bases, and ribose ordeoxyribose sugar linked by phosphodiester bonds.

In some embodiments, the cargo includes or is composed of nucleotideanalogs that have been chemically modified to improve stability,half-life, or specificity or affinity for a target receptor, relative toa DNA or RNA counterpart. The chemical modifications include chemicalmodification of nucleobases, sugar moieties, nucleotide linkages, orcombinations thereof. As used herein ‘modified nucleotide” or“chemically modified nucleotide” defines a nucleotide that has achemical modification of one or more of the heterocyclic base, sugarmoiety or phosphate moiety constituents. In some embodiments, the chargeof the modified nucleotide is reduced compared to DNA or RNA of the samenucleobase sequence. For example, the oligonucleotide can have lownegative charge, no charge, or positive charge.

Typically, nucleoside analogs support bases capable of hydrogen bondingby Watson-Crick base pairing to standard polynucleotide bases, where theanalog backbone presents the bases in a manner to permit such hydrogenbonding in a sequence-specific fashion between the oligonucleotideanalog molecule and bases in a standard polynucleotide (e.g.,single-stranded RNA or single-stranded DNA). In some embodiments, theanalogs have a substantially uncharged, phosphorus containing backbone.

a. Heterocyclic Bases

The principal naturally-occurring nucleotides include uracil, thymine,cytosine, adenine and guanine as the heterocyclic bases. The cargo caninclude chemical modifications to their nucleobase constituents.Chemical modifications of heterocyclic bases or heterocyclic baseanalogs may be effective to increase the binding affinity or stabilityin binding a target sequence. Chemically-modified heterocyclic basesinclude, but are not limited to, inosine, 5-(1-propynyl) uracil (pU),5-(1-propynyl) cytosine (pC), 5-methylcytosine, 8-oxo-adenine,pseudocytosine, pseudoisocytosine, 5 and2-amino-5-(2′-deoxy-.beta.-D-ribofuranosyl)pyridine (2-aminopyridine),and various pyrrolo- and pyrazolopyrimidine derivatives.

b. Sugar Modifications

Cargo can also contain nucleotides with modified sugar moieties or sugarmoiety analogs. Sugar moiety modifications include, but are not limitedto, 2′-O-aminoetoxy, 2′-O-amonioethyl (2′-OAE), 2′-O-methoxy,2′-O-methyl, 2-guanidoethyl (2′-OGE), 2′-0,4′-C-methylene (LNA),2′-O-(methoxyethyl) (2′-OME) and 2′-O-(N-(methyl)acetamido) (2′-OMA). 2′aminoethyl sugar moiety substitutions are especially preferred becausethey are protonated at neutral pH and thus suppress the charge repulsionbetween the TFO and the target duplex. This modification stabilizes theC3′-endo conformation of the ribose or dexyribose and also forms abridge with the i-1 phosphate in the purine strand of the duplex.

In some embodiments, the nucleic acid is a morpholino oligonucleotide.Morpholino oligonucleotides are typically composed of two moremorpholino monomers containing purine or pyrimidine base-pairingmoieties effective to bind, by base-specific hydrogen bonding, to a basein a polynucleotide, which are linked together by phosphorus-containinglinkages, one to three atoms long, joining the morpholino nitrogen ofone monomer to the 5′ exocyclic carbon of an adjacent monomer. Thepurine or pyrimidine base-pairing moiety is typically adenine, cytosine,guanine, uracil or thymine. The synthesis, structures, and bindingcharacteristics of morpholino oligomers are detailed in U.S. Pat. Nos.5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and5,506,337.

Important properties of the morpholino-based subunits typically include:the ability to be linked in a oligomeric form by stable, unchargedbackbone linkages; the ability to support a nucleotide base (e.g.adenine, cytosine, guanine, thymidine, uracil or inosine) such that thepolymer formed can hybridize with a complementary-base target nucleicacid, including target RNA, with high T_(m), even with oligomers asshort as 10-14 bases; the ability of the oligomer to be activelytransported into mammalian cells; and the ability of an oligomer:RNAheteroduplex to resist RNAse degradation.

In some embodiments, oligonucleotides employ morpholino-based subunitsbearing base-pairing moieties, joined by uncharged linkages, asdescribed above.

c. Internucleotide Linkages

Oligonucleotides are connected by an internucleotide bond that refers toa chemical linkage between two nucleoside moieties. Modifications to thephosphate backbone of DNA or RNA oligonucleotides may increase thebinding affinity or stability oligonucleotides, or reduce thesusceptibility of oligonucleotides nuclease digestion. Cationicmodifications, including, but not limited to, diethyl-ethylenediamide(DEED) or dimethyl-aminopropylamine (DMAP) may be especially useful dueto decrease electrostatic repulsion between the oligonucleotide and atarget. Modifications of the phosphate backbone may also include thesubstitution of a sulfur atom for one of the non-bridging oxygens in thephosphodiester linkage. This substitution creates a phosphorothioateinternucleoside linkage in place of the phosphodiester linkage.Oligonucleotides containing phosphorothioate internucleoside linkageshave been shown to be more stable in vivo.

Examples of modified nucleotides with reduced charge include modifiedinternucleotide linkages such as phosphate analogs having achiral anduncharged intersubunit linkages (e.g., Sterchak, E. P. et al., Organic.Chem., 52:4202, (1987)), and uncharged morpholino-based polymers havingachiral intersubunit linkages (see, e.g., U.S. Pat. No. 5,034,506), asdiscussed above. Some internucleotide linkage analogs includemorpholidate, acetal, and polyamide-linked heterocycles.

In another embodiment, the cargo are composed of locked nucleic acids.Locked nucleic acids (LNA) are modified RNA nucleotides (see, forexample, Braasch, et al., Chem. Biol., 8(1):1-7 (2001)). LNAs formhybrids with DNA which are more stable than DNA/DNA hybrids, a propertysimilar to that of peptide nucleic acid (PNA)/DNA hybrids. Therefore,LNA can be used just as PNA molecules would be. LNA binding efficiencycan be increased in some embodiments by adding positive charges to it.Commercial nucleic acid synthesizers and standard phosphoramiditechemistry are used to make LNAs.

In some embodiments, the cargo are composed of peptide nucleic acids.Peptide nucleic acids (PNAs) are synthetic DNA mimics in which thephosphate backbone of the oligonucleotide is replaced in its entirety byrepeating N-(2-aminoethyl)-glycine units and phosphodiester bonds aretypically replaced by peptide bonds. The various heterocyclic bases arelinked to the backbone by methylene carbonyl bonds. PNAs maintainspacing of heterocyclic bases that is similar to conventional DNAoligonucleotides, but are achiral and neutrally charged molecules.Peptide nucleic acids are composed of peptide nucleic acid monomers.

Other backbone modifications include peptide and amino acid variationsand modifications. Thus, the backbone constituents of oligonucleotidessuch as PNA may be peptide linkages, or alternatively, they may benon-peptide peptide linkages. Examples include acetyl caps, aminospacers such as 8-amino-3,6-dioxaoctanoic acid (referred to herein as0-linkers), amino acids such as lysine are particularly useful ifpositive charges are desired in the PNA, and the like. Methods for thechemical assembly of PNAs are well known. See, for example, U.S. Pat.Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571and 5,786,571.

Cargo optionally include one or more terminal residues or modificationsat either or both termini to increase stability, and/or affinity of theoligonucleotide for its target. Commonly used positively chargedmoieties include the amino acids lysine and arginine, although otherpositively charged moieties may also be useful. Cargo may further bemodified to be end capped to prevent degradation using a propylaminegroup. Procedures for 3′ or 5′ capping oligonucleotides are well knownin the art.

In some embodiments, the nucleic acid can be single stranded or doublestranded.

C. Pharmaceutical Compositions

The compositions can be used therapeutically in combination with apharmaceutically acceptable carrier.

The compositions including nucleic acid cargo complexed with 3E10antibody are preferably employed for therapeutic uses in combinationwith a suitable pharmaceutical carrier. Such compositions include aneffective amount of the composition, and a pharmaceutically acceptablecarrier or excipient.

The compositions may be in a formulation for administration topically,locally or systemically in a suitable pharmaceutical carrier.Remington's Pharmaceutical Sciences, 15th Edition by E. W. Martin (MarkPublishing Company, 1975), discloses typical carriers and methods ofpreparation. The complexes may also be encapsulated in suitablebiocompatible particles formed of biodegradable or non-biodegradablepolymers or proteins or liposomes for targeting to cells. Such systemsare well known to those skilled in the art. In some embodiments, thecomplexes are encapsulated in nanoparticles.

Formulations for injection may be presented in unit dosage form, e.g.,in ampules or in multi-dose containers, optionally with an addedpreservative. The compositions may take such forms as sterile aqueous ornonaqueous solutions, suspensions and emulsions, which can be isotonicwith the blood of the subject in certain embodiments. Examples ofnonaqueous solvents are polypropylene glycol, polyethylene glycol,vegetable oil such as olive oil, sesame oil, coconut oil, arachis oil,peanut oil, mineral oil, injectable organic esters such as ethyl oleate,or fixed oils including synthetic mono or di-glycerides. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, 1,3-butandiol, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers, andelectrolyte replenishers (such as those based on Ringer's dextrose). Thematerials may be in solution, emulsions, or suspension (for example,incorporated into particles, liposomes, or cells). Typically, anappropriate amount of a pharmaceutically-acceptable salt is used in theformulation to render the formulation isotonic. Trehalose, typically inthe amount of 1-5%, may be added to the pharmaceutical compositions. ThepH of the solution is preferably from about 5 to about 8, and morepreferably from about 7 to about 7.5.

Pharmaceutical compositions may include carriers, thickeners, diluents,buffers, preservatives, and surface-active agents. Carrier formulationcan be found in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa. Those of skill in the art can readily determine thevarious parameters for preparing and formulating the compositionswithout resort to undue experimentation.

The compositions alone or in combination with other suitable components,can also be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and air. For administrationby inhalation, the compounds are delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant.

In some embodiments, the include pharmaceutically acceptable carrierswith formulation ingredients such as salts, carriers, buffering agents,emulsifiers, diluents, excipients, chelating agents, preservatives,solubilizers, or stabilizers.

The nucleic acids may be conjugated to lipophilic groups likecholesterol and lauric and lithocholic acid derivatives with C32functionality to improve cellular uptake. For example, cholesterol hasbeen demonstrated to enhance uptake and serum stability of siRNA invitro (Lorenz, et al., Bioorg. Med. Chem. Lett., 14(19):4975-4977(2004)) and in vivo (Soutschek, et al., Nature, 432(7014):173-178(2004)). In addition, it has been shown that binding of steroidconjugated oligonucleotides to different lipoproteins in thebloodstream, such as LDL, protect integrity and facilitatebiodistribution (Rump, et al., Biochem. Pharmacol., 59(11):1407-1416(2000)). Other groups that can be attached or conjugated to the nucleicacids described above to increase cellular uptake, include acridinederivatives; cross-linkers such as psoralen derivatives, azidophenacyl,proflavin, and azidoproflavin; artificial endonucleases; metal complexessuch as EDTA-Fe(II) and porphyrin-Fe(II); alkylating moieties; nucleasessuch as alkaline phosphatase; terminal transferases; abzymes;cholesteryl moieties; lipophilic carriers; peptide conjugates; longchain alcohols; phosphate esters; radioactive markers; non-radioactivemarkers; carbohydrates; and polylysine or other polyamines U.S. Pat. No.6,919,208 to Levy, et al., also describes methods for enhanced delivery.These pharmaceutical formulations may be manufactured in a manner thatis itself known, e.g., by means of conventional mixing, dissolving,granulating, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes.

Further carriers include sustained release preparations such assemi-permeable matrices of solid hydrophobic polymers containing thecomplexes, which matrices are in the form of shaped particles, e.g.,films, liposomes or microparticles. Implantation includes insertingimplantable drug delivery systems, e.g., microspheres, hydrogels,polymeric reservoirs, cholesterol matrixes, polymeric systems, e.g.,matrix erosion and/or diffusion systems and non-polymeric systems.Inhalation includes administering the composition with an aerosol in aninhaler, either alone or attached to a carrier that can be absorbed. Forsystemic administration, it may be preferred that the composition isencapsulated in liposomes.

The compositions may be delivered in a manner which enablestissue-specific uptake of the agent and/or nucleotide delivery system,using invasive devices such as vascular or urinary catheters, and usinginterventional devices such as stents having drug delivery capabilityand configured as expansive devices or stent grafts.

The formulations may be delivered using a bioerodible implant by way ofdiffusion or by degradation of the polymeric matrix. In certainembodiments, the administration of the formulation may be designed toresult in sequential exposures to the composition, over a certain timeperiod, for example, hours, days, weeks, months or years. This may beaccomplished, for example, by repeated administrations of a formulationor by a sustained or controlled release delivery system in which thecompositions are delivered over a prolonged period without repeatedadministrations.

Other delivery systems suitable include time-release, delayed release,sustained release, or controlled release delivery systems. Such systemsmay avoid repeated administrations in many cases, increasing convenienceto the subject and the physician. Many types of release delivery systemsare available and known to those of ordinary skill in the art. Theyinclude, for example, polymer-based systems such as polylactic and/orpolyglycolic acids, polyanhydrides, polycaprolactones, copolyoxalates,polyesteramides, polyorthoesters, polyhydroxybutyric acid, and/orcombinations of these. Microcapsules of the foregoing polymerscontaining nucleic acids are described in, for example, U.S. Pat. No.5,075,109. Other examples include non-polymer systems that arelipid-based including sterols such as cholesterol, cholesterol esters,and fatty acids or neutral fats such as mono-, di- and triglycerides;hydrogel release systems; liposome-based systems; phospholipidbased-systems; silastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; orpartially fused implants. The formulation may be as, for example,microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, orpolymeric systems. In some embodiments, the system may allow sustainedor controlled release of the composition to occur, for example, throughcontrol of the diffusion or erosion/degradation rate of the formulationscontaining the complexes.

Complexes include nucleic acid cargo and antibody, and compositionsthereof can be formulated for pulmonary or mucosal administration. Theadministration can include delivery of the composition to the lungs,nasal, oral (sublingual, buccal), vaginal, or rectal mucosa. The termaerosol as used herein refers to any preparation of a fine mist ofparticles, which can be in solution or a suspension, whether or not itis produced using a propellant. Aerosols can be produced using standardtechniques, such as ultrasonication or high-pressure treatment.

For administration via the upper respiratory tract, the formulation canbe formulated into a solution, e.g., water or isotonic saline, bufferedor un-buffered, or as a suspension, for intranasal administration asdrops or as a spray. Preferably, such solutions or suspensions areisotonic relative to nasal secretions and of about the same pH, ranginge.g., from about pH 4.0 to about pH 7.4 or, from pH 6.0 to pH 7.0.Buffers should be physiologically compatible and include, simply by wayof example, phosphate buffers.

The complexes can be delivered to the target cells using a particledelivery vehicle. Nanoparticles generally refers to particles in therange of between 500 nm to less than 0.5 nm, preferably having adiameter that is between 50 and 500 nm, more preferably having adiameter that is between 50 and 300 nm. Cellular internalization ofpolymeric particles is highly dependent upon their size, withnanoparticulate polymeric particles being internalized by cells withmuch higher efficiency than micoparticulate polymeric particles. Forexample, Desai, et al. have demonstrated that about 2.5 times morenanoparticles that are 100 nm in diameter are taken up by culturedCaco-2 cells as compared to microparticles having a diameter on 1 μM(Desai, et al., Pharm. Res., 14:1568-73 (1997)). Nanoparticles also havea greater ability to diffuse deeper into tissues in vivo.

In some embodiments, the delivery vehicle is a dendrimer.

Examples of preferred biodegradable polymers include synthetic polymersthat degrade by hydrolysis such as poly(hydroxy acids), such as polymersand copolymers of lactic acid and glycolic acid, other degradablepolyesters, polyanhydrides, poly(ortho)esters, polyesters,polyurethanes, poly(butic acid), poly(valeric acid), poly(caprolactone),poly(hydroxyalkanoates), poly(lactide-co-caprolactone), andpoly(amine-co-ester) polymers, such as those described in Zhou, et al.,Nature Materials, 11:82-90 (2012) and WO 2013/082529, U.S. PublishedApplication No. 2014/0342003, and PCT/US2015/061375.

In some embodiments, particularly those for targeting T cell in vivo,for example, for in vivo production of CAR T cells, immune cell or Tcell markers such as CD3, CD7, or CD8, or markers of a target tissuesuch as the liver, can be targeted. For example, anti-CD8 antibodies andanti-CD3 Fab fragments have both been used to target T cells in vivo(Pfeiffer, et al., EMBO Mol Med., 10(11) (2018). pii: e9158. doi:10.15252/emmm 201809158., Smith, et al., Nat Nanotechnol., 12(8):813-820(2017). doi: 10.1038/nnano 2017.57). Thus, in some embodiments, theparticle or other delivery vehicle includes a targeting moiety specificfor CD3, CD7, CD8, or another immune cell (e.g., T cell) marker, or amarker for a specific tissue such as the thymus, spleen, or liver. Thebinding moiety can be, for example, an antibody or antigen bindingfragment thereof.

Targeting moieties can be associated with, linked, conjugated, orotherwise attached directly or indirectly to a nanoparticle or otherdelivery vehicle thereof. Targeting molecules can be proteins, peptides,nucleic acid molecules, saccharides or polysaccharides that bind to areceptor or other molecule on the surface of a targeted cell. The degreeof specificity and the avidity of binding to the graft can be modulatedthrough the selection of the targeting molecule.

Examples of moieties include, for example, targeting moieties whichprovide for the delivery of molecules to specific cells, e.g.,antibodies to hematopoietic stem cells, CD34⁺ cells, T cells or anyother preferred cell type, as well as receptor and ligands expressed onthe preferred cell type. Preferably, the moieties target hematopoeiticstem cells. Examples of molecules targeting extracellular matrix (“ECM”)include glycosaminoglycan (“GAG”) and collagen. In one embodiment, theexternal surface of polymer particles may be modified to enhance theability of the particles to interact with selected cells or tissue. Themethod described above wherein an adaptor element conjugated to atargeting molecule is inserted into the particle is preferred. However,in another embodiment, the outer surface of a polymer micro- ornanoparticle having a carboxy terminus may be linked to targetingmolecules that have a free amine terminus.

Other useful ligands attached to polymeric micro- and nanoparticlesinclude pathogen-associated molecular patterns (PAMPs). PAMPs targetToll-like Receptors (TLRs) on the surface of the cells or tissue, orsignal the cells or tissue internally, thereby potentially increasinguptake. PAMPs conjugated to the particle surface or co-encapsulated mayinclude: unmethylated CpG DNA (bacterial), double-stranded RNA (viral),lipopolysacharride (bacterial), peptidoglycan (bacterial),lipoarabinomannin (bacterial), zymosan (yeast), mycoplasmal lipoproteinssuch as MALP-2 (bacterial), flagellin (bacterial)poly(inosinic-cytidylic) acid (bacterial), lipoteichoic acid (bacterial)or imidazoquinolines (synthetic).

In another embodiment, the outer surface of the particle may be treatedusing a mannose amine, thereby mannosylating the outer surface of theparticle. This treatment may cause the particle to bind to the targetcell or tissue at a mannose receptor on the antigen presenting cellsurface. Alternatively, surface conjugation with an immunoglobulinmolecule containing an Fc portion (targeting Fc receptor), heat shockprotein moiety (HSP receptor), phosphatidylserine (scavenger receptors),and lipopolysaccharide (LPS) are additional receptor targets on cells ortissue.

Lectins that can be covalently attached to micro- and nanoparticles torender them target specific to the mucin and mucosal cell layer.

The choice of targeting moiety will depend on the method ofadministration of the nanoparticle composition and the cells or tissuesto be targeted. The targeting molecule may generally increase thebinding affinity of the particles for cell or tissues or may target thenanoparticle to a particular tissue in an organ or a particular celltype in a tissue. In some embodiments, the targeting moiety targets thethymus, spleen, or cancer cells

The covalent attachment of any of the natural components of mucin ineither pure or partially purified form to the particles would decreasethe surface tension of the bead-gut interface and increase thesolubility of the bead in the mucin layer. The attachment of polyaminoacids containing extra pendant carboxylic acid side groups, e.g.,polyaspartic acid and polyglutamic acid, increases bioadhesiveness.Using polyamino acids in the 15,000 to 50,000 kDa molecular weight rangeyields chains of 120 to 425 amino acid residues attached to the surfaceof the particles. The polyamino chains increase bioadhesion by means ofchain entanglement in mucin strands as well as by increased carboxyliccharge.

III. Methods of Use

Methods for using 3E10 to enhance delivery of nucleic acid constructsare provided. Typically an effective amount of 3E10 antibody is firstcontacted with a nucleic acid cargo whose delivery into cells isdesired. For example, the nucleic acid cargo and antibody can be mixedin solution for sufficient time for the nucleic acid cargo and antibodyto form complexes. Next, the mixture is contacted with cells. In otherembodiments, the cargo and antibody are added to a solution containingor otherwise bathing cells, and the complexes are formed in the presenceof the cells. The complexes can be contacted with cells in vitro, exvivo, or in vivo. Thus, in some embodiments, the solution of complexesis added to the cells in culture or injected into an animal to betreated.

It is believed that the antibody helps deliver the nucleic acid intocell nuclei, and then alters the function of the RAD51 pathway which canpromote gene editing by the donor DNA. The approach has no sequencelimitations to the design of the nucleic acid cargo. The treatment canbe, for example, administration of a mixture of an antibody and nucleicacid cargo to a subject in need thereof by simple IV administration

The compositions and methods can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more different nucleic acid constructs formed of RNA, DNA, PNA orother modified nucleic acids, or a combination thereof.

The effective amount or therapeutically effective amount of thecomposition can be a dosage sufficient to treat, inhibit, or alleviateone or more symptoms of a disease or disorder, or to otherwise provide adesired pharmacologic and/or physiologic effect, for example, reducing,inhibiting, or reversing one or more of the pathophysiologicalmechanisms underlying a disease or disorder.

An effective amount may also be an amount effective to increase therate, quantity, and/or quality of delivery of the nucleic acid cargorelative to administration of the cargo in the absence of the antibody.The formulation of the composition is made to suit the mode ofadministration. Pharmaceutically acceptable carriers are determined inpart by the particular composition being administered, as well as by theparticular method used to administer the composition. Accordingly, thereis a wide variety of suitable formulations of pharmaceuticalcompositions containing the complexes. The precise dosage will varyaccording to a variety of factors such as subject-dependent variables(e.g., age, immune system health, clinical symptoms etc.).

The composition can be administered or otherwise contacted with targetcells once, twice, or three times daily; one, two, three, four, five,six, seven times a week, one, two, three, four, five, six, seven oreight times a month. For example, in some embodiments, the compositionis administered every two or three days, or on average about 2 to about4 times about week. Thus, in some embodiments, the composition isadministered as part of dosage regimen including two or more separatetreatments.

Dosage regimens include maintenance regimens, where the dosage remainsthe same between two or more administrations, escalation regimens wherethe dosage increases between two or more administrations, de-escalationregimens, where the dosage decreases between two or moreadministrations, or a combination thereof.

In some embodiments, the first dose can be a low dose. Dose escalationcan be continued until a satisfactory biochemical or clinical responseis reached. The clinical response will depend on the disease or disorderbeing treated, and/or the desired outcome. In some embodiments thedosage may increase until a therapeutic effect is identified, preferablywithout also inducing undesired toxicity or an acceptably high amountthereof. Next, the dosages can be maintained or steadily reduced to amaintenance dose. The methods can used to standardize, optimize, orcustomize the dose level, dose frequency, or duration of the therapy.

Generally, prior to administration, particularly for in vivoadministration, antibody and nucleic acid are mixed for a period of timeat room temperature. In some embodiments, time of complexation rangesfrom, for example, 1 minute to 30 minutes, or 10 minutes to 20, eachinclusive, with a preferred complexation time of about 15 minutes.Antibody dose can range from 0.0001 mg to 1 mg, each inclusive, with apreferred dose of about 0.1 mg. Nucleic acid dose can range from 0.001μg to 100 μg, inclusive, with a preferred dose of 10 μg. The in vivodata below (e.g., FIG. 6 B) was produced using 0.1 mg 3E10, 10 μg ofmRNA, and complexed for 15 minutes.

The Examples below may indicate that DNA cargo may be delivered moregenerally to multiple tissues and not restricted to tumors, while RNAdelivery may be more selective for tumor tissue. Thus, in someembodiments, RNA cargo (e.g., alone) may be selectively delivered tocancer cells or other tumor tissues. In some embodiments, when widerdistribution of RNA cargo is desired, the RNA may be mixed with DNA(e.g., carrier DNA) to facilitate delivery to non-cancer/tumor tissues.Carrier DNA can be, for example, plasmid DNA or low molecular weight,from e.g., salmon sperm. In some embodiments, carrier DNA is non-codingDNA. Carrier DNA can be single stranded or double stranded or acombination thereof. In some embodiments, carrier DNA is composed ofnucleic acids having 1-10, 1-100, 1-1,000, or 1-10,000 nucleotides inlength, or any subrange or integer thereof, or combination thereof.Typically carrier DNA is not conjugated or otherwise covalently attachedto the antibody. Typically carrier DNA is co-incubated with cargonucleic acid (e.g., RNA) and antibody, and co-delivered as a complextherewith. In some embodiments, the carrier DNA is non-coding DNA.

A. In vitro and Ex vivo Methods

For in vitro and ex vivo methods, cells are typically contacted with thecomposition while in culture. For ex vivo methods, cells may be isolatedfrom a subject and contacted ex vivo with the composition to producecells containing the cargo nucleic acid(s). In a preferred embodiment,the cells are isolated from the subject to be treated or from asyngeneic host. Target cells can be removed from a subject prior tocontacting with composition.

B. In vivo Methods

In some embodiments, in vivo delivery of nucleic acid cargo to cells isused for gene editing and/or treatment of a disease or disorder in asubject. The composition, typically including antibody-nucleic acidcargo, can be administered directly to a subject for in vivo therapy.

In general, methods of administering compounds, including antibodies,oligonucleotides and related molecules, are well known in the art. Inparticular, the routes of administration already in use for nucleic acidtherapeutics, along with formulations in current use, provide preferredroutes of administration and formulation for the donor oligonucleotidesdescribed above. Preferably the composition is injected or infused intothe animal.

The compositions can be administered by a number of routes including,but not limited to, intravenous, intraperitoneal, intraamniotic,intramuscular, subcutaneous, or topical (sublingual, rectal, intranasal,pulmonary, rectal mucosa, and vaginal), and oral (sublingual, buccal).

In some embodiments, the composition is formulated for pulmonarydelivery, such as intranasal administration or oral inhalation.Administration of the formulations may be accomplished by any acceptablemethod that allows the complexes to reach their targets. Theadministration may be localized (i.e., to a particular region,physiological system, tissue, organ, or cell type) or systemic,depending on the condition being treated. Compositions and methods forin vivo delivery are also discussed in WO 2017/143042.

The methods can also include administering an effective amount of theantibody-nucleic acid complex composition to an embryo or fetus, or thepregnant mother thereof, in vivo. In some methods, compositions aredelivered in utero by injecting and/or infusing the compositions into avein or artery, such as the vitelline vein or the umbilical vein, orinto the amniotic sac of an embryo or fetus. See, e.g., Ricciardi, etal., Nat Commun. 2018 Jun. 26; 9(1):2481. doi:10.1038/s41467-018-04894-2, and WO 2018/187493.

C. Applications

Nucleic acid cargo, e.g., mRNA, functional nucleic acid, DNA expressionconstructs, vectors, etc., encoding a polypeptide of interest orfunctional nucleic acid, can be delivered into cells using a 3E10antibody, for expression of, or inhibition of, a polypeptide in thecells. The compositions and methods can be used over a range ofdifferent applications. Non-limiting examples include CRISPR and gRNAexpression vectors +/− editing DNAs, delivery of large DNAs (plasmidsand expression vectors), gene replacement and gene therapy, delivery ofDNAs and/or RNAs to, for example, generate CAR-T cells in vivo or exvivo and to simplify CAR-T cell production in vivo or ex vivo, deliveryof siRNAs, delivery of mRNAs, etc. Exemplary applications related togene therapy/gene editing and immunomodulation, particularly chimericantigen receptor T cell production, are discussed below.

1. Gene Therapy and Editing

In some embodiments, the compositions are used for gene editing.

For example, the methods can be especially useful to treat geneticdeficiencies, disorders and diseases caused by mutations in singlegenes, for example, to correct genetic deficiencies, disorders anddiseases caused by point mutations. If the target gene contains amutation that is the cause of a genetic disorder, then the methods canbe used for mutagenic repair that may restore the DNA sequence of thetarget gene to normal. The target sequence can be within the coding DNAsequence of the gene or within an intron. The target sequence can alsobe within DNA sequences that regulate expression of the target gene,including promoter or enhancer sequences.

In the methods herein, cells that have been contacted with the complexesmay be administered to a subject. The subject may have a disease ordisorder such as hemophilia, muscular dystrophy, globinopathies, cysticfibrosis, xeroderma pigmentosum, or lysosomal storage diseases. In suchembodiments, gene modification, gene replacement, gene addition, or acombination thereof, may occur in an effective amount to reduce one ormore symptoms of the disease or disorder in the subject.

In some embodiments, the DNA cargo includes a nucleic acid encoding anuclease, a donor oligonucleotide or nucleic acid encoding a donoroligonucleotide, or a combination thereof.

a. Gene Editing Nuclease

Nucleic acid cargos include those that encode an element or elementsthat induce a single or a double strand break in the target cell'sgenome, and optionally, but preferable in combination with otherelements such as donor oligonucleotides and/or, particularly in the caseof CRISPR/Cas, other elements of the system such as gRNA. Thecompositions can be used, for example, to reduce or otherwise modifyexpression of a target gene.

i. Strand Break Inducing Elements CRISPR/Cas

In some embodiments, the nucleic acid cargo includes one or moreelements of a CRISPR/Cas-mediated genome editing composition, a nucleicacid encoding one or more elements of a CRISPR/Cas-mediated genomeediting composition, or a combination thereof. As used herein,CRISPR/Cas-mediated genome editing composition refers to the elements ofa CRISPR system needed to carry out CRISPR/Cas-mediated genome editingin a mammalian subject. As discussed in more detail below,CRISPR/Cas-mediated genome editing compositions typically include one ormore nucleic acids encoding a crRNA, a tracrRNA (or chimeric thereofalso referred to a guide RNA or single guide RNA) and a Cas enzyme, suchas Cas9. The CRISPR/Cas-mediated genome editing composition canoptionally include a donor polynucleotide that can be recombined intothe target cell's genome at or adjacent to the target site (e.g., thesite of single or double stand break induced by the Cas9).

The CRISPR/Cas system has been adapted for use as gene editing(silencing, enhancing or changing specific genes) for use in eukaryotes(see, for example, Cong, Science, 15:339(6121):819-823 (2013) and Jinek,et al., Science, 337(6096):816-21 (2012)). By transfecting a cell withthe required elements including a cas gene and specifically designedCRISPRs, the organism's genome can be cut and modified at any desiredlocation. Methods of preparing compositions for use in genome editingusing the CRISPR/Cas systems are described in detail in WO 2013/176772and WO 2014/018423, which are specifically incorporated by referenceherein in their entireties.

The methods of delivery disclosed herein are suitable for use withnumerous variations on the CRISPR/Cas system.

In general, “CRISPR system” refers collectively to transcripts and otherelements involved in the expression of or directing the activity ofCRISPR-associated (“Cas”) genes, including sequences encoding a Casgene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or anactive partial tracrRNA), a tracr-mate sequence (encompassing a “directrepeat” and a tracrRNA-processed partial direct repeat in the context ofan endogenous CRISPR system), a guide sequence (also referred to as a“spacer” in the context of an endogenous CRISPR system), or othersequences and transcripts from a CRISPR locus. One or more tracr matesequences operably linked to a guide sequence (e.g., directrepeat-spacer-direct repeat) can also be referred to as pre-crRNA(pre-CRISPR RNA) before processing or crRNA after processing by anuclease.

As discussed in more detail below, in some embodiments, a tracrRNA andcrRNA are linked and form a chimeric crRNA-tracrRNA hybrid where amature crRNA is fused to a partial tracrRNA via a synthetic stem loop tomimic the natural crRNA:tracrRNA duplex as described in Cong, Science,15:339(6121):819-823 (2013) and Jinek, et al., Science, 337(6096):816-21(2012)). A single fused crRNA-tracrRNA construct is also referred toherein as a guide RNA or gRNA (or single-guide RNA (sgRNA)). Within ansgRNA, the crRNA portion can be identified as the ‘target sequence’ andthe tracrRNA is often referred to as the ‘scaffold’.

In some embodiments, one or more elements of a CRISPR system is derivedfrom a type I, type II, or type III CRISPR system. In some embodiments,one or more elements of a CRISPR system is derived from a particularorganism including an endogenous CRISPR system, such as Streptococcuspyogenes.

In general, a CRISPR system is characterized by elements that promotethe formation of a CRISPR complex at the site of a target sequence (alsoreferred to as a protospacer in the context of an endogenous CRISPRsystem). In the context of formation of a CRISPR complex, “targetsequence” refers to a sequence to which a guide sequence is designed tohave complementarity, where hybridization between a target sequence anda guide sequence promotes the formation of a CRISPR complex. A targetsequence can be any polynucleotide, such as DNA or RNA polynucleotides.In some embodiments, a target sequence is located in the nucleus orcytoplasm of a cell.

In the target nucleic acid, each protospacer is associated with aprotospacer adjacent motif (PAM) whose recognition is specific toindividual CRISPR systems. In the Streptococcus pyogenes CRISPR/Cassystem, the PAM is the nucleotide sequence NGG. In the Streptococcusthermophiles CRISPR/Cas system, the PAM is the nucleotide sequence isNNAGAAW. The tracrRNA duplex directs Cas to the DNA target consisting ofthe protospacer and the requisite PAM via heteroduplex formation betweenthe spacer region of the crRNA and the protospacer DNA.

Typically, in the context of an endogenous CRISPR system, formation of aCRISPR complex (including a guide sequence hybridized to a targetsequence and complexed with one or more Cas proteins) results incleavage of one or both strands in or near (e.g., within 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence.All or a portion of the tracr sequence may also form part of a CRISPRcomplex, such as by hybridization to all or a portion of a tracr matesequence that is operably linked to the guide sequence.

There are many resources available for helping practitioners determinesuitable target sites once a desired DNA target sequence is identified.For example, numerous public resources, including a bioinformaticallygenerated list of about 190,000 potential sgRNAs, targeting more than40% of human exons, are available to aid practitioners in selectingtarget sites and designing the associate sgRNA to affect a nick ordouble strand break at the site. See also, crispr.u-psud.fr/, a tooldesigned to help scientists find CRISPR targeting sites in a wide rangeof species and generate the appropriate crRNA sequence.

In some embodiments, one or more vectors driving expression of one ormore elements of a CRISPR system are introduced into a target cell suchthat expression of the elements of the CRISPR system direct formation ofa CRISPR complex at one or more target sites. For example, a Cas enzyme,a guide sequence linked to a tracr-mate sequence, and a tracr sequencecould each be operably linked to separate regulatory elements onseparate vectors. Alternatively, two or more of the elements expressedfrom the same or different regulatory elements may be combined in asingle vector, with one or more additional vectors providing anycomponents of the CRISPR system not included in the first vector. CRISPRsystem elements that are combined in a single vector may be arranged inany suitable orientation, such as one element located 5′ with respect to(“upstream” of) or 3′ with respect to (“downstream” of) a secondelement. The coding sequence of one element can be located on the sameor opposite strand of the coding sequence of a second element, andoriented in the same or opposite direction. In some embodiments, asingle promoter drives expression of a transcript encoding a CRISPRenzyme and one or more of the guide sequence, tracr mate sequence(optionally operably linked to the guide sequence), and a tracr sequenceembedded within one or more intron sequences (e.g., each in a differentintron, two or more in at least one intron, or all in a single intron).In some embodiments, the CRISPR enzyme, guide sequence, tracr matesequence, and tracr sequence are operably linked to and expressed fromthe same promoter.

In some embodiments, a vector includes one or more insertion sites, suchas a restriction endonuclease recognition sequence (also referred to asa “cloning site”). In some embodiments, one or more insertion sites(e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moreinsertion sites) are located upstream and/or downstream of one or moresequence elements of one or more vectors. In some embodiments, a vectorincludes an insertion site upstream of a tracr mate sequence, andoptionally downstream of a regulatory element operably linked to thetracr mate sequence, such that following insertion of a guide sequenceinto the insertion site and upon expression the guide sequence directssequence-specific binding of a CRISPR complex to a target sequence in aeukaryotic cell. In some embodiments, a vector includes two or moreinsertion sites, each insertion site being located between two tracrmate sequences so as to allow insertion of a guide sequence at eachsite. In such an arrangement, the two or more guide sequences caninclude two or more copies of a single guide sequence, two or moredifferent guide sequences, or combinations of these. When multipledifferent guide sequences are used, a single expression construct may beused to target CRISPR activity to multiple different, correspondingtarget sequences within a cell. For example, a single vector can includeabout or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 guidesequences. In some embodiments, about or more than about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, such guide-sequence-containing vectors may be provided,and optionally delivered to a cell.

In some embodiments, a vector includes a regulatory element operablylinked to an enzyme-coding sequence encoding a CRISPR enzyme, such as aCas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B,Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 andCsx12), Cas1O, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2,Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2,Csb3, Csx17, Csx14, Csx1O, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2,Csf3, Csf4, homologues thereof, or modified versions thereof. In someembodiments, the unmodified CRISPR enzyme has DNA cleavage activity,such as Cas9. In some embodiments, the CRISPR enzyme directs cleavage ofone or both strands at the location of a target sequence, such as withinthe target sequence and/or within the complement of the target sequence.In some embodiments, the CRISPR enzyme directs cleavage of one or bothstrands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100,200, 500, or more base pairs from the first or last nucleotide of atarget sequence.

In some embodiments, a vector encodes a CRISPR enzyme that is mutatedwith respect to a corresponding wild-type enzyme such that the mutatedCRISPR enzyme lacks the ability to cleave one or both strands of atarget polynucleotide containing a target sequence. For example, anaspartate-to-alanine substitution (D10A) in the RuvC I catalytic domainof Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves bothstrands to a nickase (cleaves a single strand). Other examples ofmutations that render Cas9 a nickase include, without limitation, H840A,N854A, and N863A. As a further example, two or more catalytic domains ofCas9 (RuvC I, RuvC II, and RuvC III) can be mutated to produce a mutatedCas9 substantially lacking all DNA cleavage activity. In someembodiments, a D10A mutation is combined with one or more of H840A,N854A, or N863A mutations to produce a Cas9 enzyme substantially lackingall DNA cleavage activity. In some embodiments, a CRISPR enzyme isconsidered to substantially lack all DNA cleavage activity when the DNAcleavage activity of the mutated enzyme is less than about 25%, 10%,5%>, 1%>, 0.1%>, 0.01%, or lower with respect to its non-mutated form.

In some embodiments, an enzyme coding sequence encoding a CRISPR enzymeis codon optimized for expression in particular cells, such aseukaryotic cells. The eukaryotic cells can be those of or derived from aparticular organism, such as a mammal, including but not limited tohuman, mouse, rat, rabbit, dog, or non-human primate. In general, codonoptimization refers to a process of modifying a nucleic acid sequencefor enhanced expression in the host cells of interest by replacing atleast one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15,20, 25, 50, or more codons) of the native sequence with codons that aremore frequently or most frequently used in the genes of that host cellwhile maintaining the native amino acid sequence. Various speciesexhibit particular bias for certain codons of a particular amino acid.Codon bias (differences in codon usage between organisms) oftencorrelates with the efficiency of translation of messenger RNA (mRNA),which is in turn believed to be dependent on, among other things, theproperties of the codons being translated and the availability ofparticular transfer RNA (tRNA) molecules.

The predominance of selected tRNAs in a cell is generally a reflectionof the codons used most frequently in peptide synthesis. Accordingly,genes can be tailored for optimal gene expression in a given organismbased on codon optimization. Codon usage tables are readily available,for example, at the “Codon Usage Database”, and these tables can beadapted in a number of ways. See Nakamura, Y., et al., Nucl. Acids Res.,28:292 (2000). Computer algorithms for codon optimizing a particularsequence for expression in a particular host cell, for example GeneForge (Aptagen; Jacobus, PA), are also available. In some embodiments,one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, orall codons) in a sequence encoding a CRISPR enzyme correspond to themost frequently used codon for a particular amino acid.

In some embodiments, a vector encodes a CRISPR enzyme including one ormore nuclear localization sequences (NLSs). When more than one NLS ispresent, each may be selected independently of the others, such that asingle NLS may be present in more than one copy and/or in combinationwith one or more other NLSs present in one or more copies. In someembodiments, an NLS is considered near the N- or C-terminus when thenearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20,25, 30, 40, 50, or more amino acids along the polypeptide chain from theN- or C-terminus.

In general, the one or more NLSs are of sufficient strength to driveaccumulation of the CRISPR enzyme in a detectable amount in the nucleusof a eukaryotic cell. In general, strength of nuclear localizationactivity may derive from the number of NLSs in the CRISPR enzyme, theparticular NLS(s) used, or a combination of these factors.

Detection of accumulation in the nucleus may be performed by anysuitable technique. For example, a detectable marker may be fused to theCRISPR enzyme, such that location within a cell may be visualized, suchas in combination with a means for detecting the location of the nucleus(e.g., a stain specific for the nucleus such as DAPI). Cell nuclei mayalso be isolated from cells, the contents of which may then be analyzedby any suitable process for detecting protein, such asimmunohistochemistry, Western blot, or enzyme activity assay.Accumulation in the nucleus may also be determined indirectly, such asby an assay for the effect of CRISPR complex formation (e.g., assay forDNA cleavage or mutation at the target sequence, or assay for alteredgene expression activity affected by CRISPR complex formation and/orCRISPR enzyme activity), as compared to a control no exposed to theCRISPR enzyme or complex, or exposed to a CRISPR enzyme lacking the oneor more NLSs.

In some embodiments, one or more of the elements of CRISPR system areunder the control of an inducible promoter, which can include inducibleCas, such as Cas9.

Cong, Science, 15:339(6121):819-823 (2013) reported heterologousexpression of Cas9, tracrRNA, pre-crRNA (or Cas9 and sgRNA) can achievetargeted cleavage of mammalian chromosomes. Therefore, CRISPR systemutilized in the methods disclosed herein, and thus the cargo nucleicacid, be a vector system which can include one or more vectors encodingelements of the CRISPR system which can include a first regulatoryelement operably linked to a CRISPR/Cas system chimeric RNA (chiRNA)polynucleotide sequence, wherein the polynucleotide sequence includes(a) a guide sequence capable of hybridizing to a target sequence in aeukaryotic cell, (b) a tracr mate sequence, and (c) a tracr sequence;and a second regulatory element operably linked to an enzyme-codingsequence encoding a CRISPR enzyme which can optionally include at leastone or more nuclear localization sequences. Elements (a), (b) and (c)can arranged in a 5′ to 3 orientation, wherein components I and II arelocated on the same or different vectors of the system, wherein whentranscribed, the tracr mate sequence hybridizes to the tracr sequenceand the guide sequence directs sequence-specific binding of a CRISPRcomplex to the target sequence, and wherein the CRISPR complex caninclude the CRISPR enzyme complexed with (1) the guide sequence that ishybridized to the target sequence, and (2) the tracr mate sequence thatis hybridized to the tracr sequence, wherein the enzyme coding sequenceencoding the CRISPR enzyme further encodes a heterologous functionaldomain. In some embodiments, one or more of the vectors also encodes asuitable Cas enzyme, for example, Cas9. The different genetic elementscan be under the control of the same or different promoters.

While the specifics can be varied in different engineered CRISPRsystems, the overall methodology is similar. A practitioner interestedin using CRISPR technology to target a DNA sequence (identified usingone of the many available online tools) can insert a short DNA fragmentcontaining the target sequence into a guide RNA expression plasmid. ThesgRNA expression plasmid contains the target sequence (about 20nucleotides), a form of the tracrRNA sequence (the scaffold) as well asa suitable promoter and necessary elements for proper processing ineukaryotic cells. Such vectors are commercially available (see, forexample, Addgene). Many of the systems rely on custom, complementaryoligos that are annealed to form a double stranded DNA and then clonedinto the sgRNA expression plasmid. Co-expression of the sgRNA and theappropriate Cas enzyme from the same or separate plasmids in transfectedcells results in a single or double strand break (depending of theactivity of the Cas enzyme) at the desired target site.

ii. Zinc Finger Nucleases

In some embodiments, the element that induces a single or a doublestrand break in the target cell's genome is a nucleic acid construct orconstructs encoding a zinc finger nucleases (ZFNs). Thus, the nucleicacid cargo can encode a ZFN.

ZFNs are typically fusion proteins that include a DNA-binding domainderived from a zinc-finger protein linked to a cleavage domain. The mostcommon cleavage domain is the Type IIS enzyme Fold. Fok1 catalyzesdouble-stranded cleavage of DNA, at 9 nucleotides from its recognitionsite on one strand and 13 nucleotides from its recognition site on theother. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and5,487,994; as well as Li et al. Proc., Natl. Acad. Sci. USA 89(1992):4275-4279; Li et al. Proc. Natl. Acad. Sci. USA, 90:2764-2768(1993); Kim et al. Proc. Natl. Acad. Sci. USA. 91:883-887 (1994a); Kimet al. J. Biol. Chem. 269:31, 978-31,982 (1994b). One or more of theseenzymes (or enzymatically functional fragments thereof) can be used as asource of cleavage domains.

The DNA-binding domain, which can, in principle, be designed to targetany genomic location of interest, can be a tandem array of Cys₂His₂ zincfingers, each of which generally recognizes three to four nucleotides inthe target DNA sequence. The Cys₂His₂ domain has a general structure:Phe (sometimes Tyr)-Cys-(2 to 4 amino acids)-Cys-(3 amino acids)-Phe(sometimes Tyr)-(5 amino acids)-Leu-(2 amino acids)-His-(3 aminoacids)-His. By linking together multiple fingers (the number varies:three to six fingers have been used per monomer in published studies),ZFN pairs can be designed to bind to genomic sequences 18-36 nucleotideslong.

Engineering methods include, but are not limited to, rational design andvarious types of empirical selection methods. Rational design includes,for example, using databases including triplet (or quadruplet)nucleotide sequences and individual zinc finger amino acid sequences, inwhich each triplet or quadruplet nucleotide sequence is associated withone or more amino acid sequences of zinc fingers which bind theparticular triplet or quadruplet sequence. See, for example, U.S. Pat.Nos. 6,140,081; 6,453,242; 6,534,261; 6,610,512; 6,746,838; 6,866,997;7,067,617; U.S. Published Application Nos. 2002/0165356; 2004/0197892;2007/0154989; 2007/0213269; and International Patent ApplicationPublication Nos. WO 98/53059 and WO 2003/016496.

iii. Transcription Activator-Like Effector Nucleases

In some embodiments, the element that induces a single or a doublestrand break in the target cell's genome is a nucleic acid construct orconstructs encoding a transcription activator-like effector nuclease(TALEN). Thus, the nucleic acid cargo can encode a TALEN.

TALENs have an overall architecture similar to that of ZFNs, with themain difference that the DNA-binding domain comes from TAL effectorproteins, transcription factors from plant pathogenic bacteria. TheDNA-binding domain of a TALEN is a tandem array of amino acid repeats,each about 34 residues long. The repeats are very similar to each other;typically they differ principally at two positions (amino acids 12 and13, called the repeat variable diresidue, or RVD). Each RVD specifiespreferential binding to one of the four possible nucleotides, meaningthat each TALEN repeat binds to a single base pair, though the NN RVD isknown to bind adenines in addition to guanine. TAL effector DNA bindingis mechanistically less well understood than that of zinc-fingerproteins, but their seemingly simpler code could prove very beneficialfor engineered-nuclease design. TALENs also cleave as dimers, haverelatively long target sequences (the shortest reported so far binds 13nucleotides per monomer) and appear to have less stringent requirementsthan ZFNs for the length of the spacer between binding sites. Monomericand dimeric TALENs can include more than 10, more than 14, more than 20,or more than 24 repeats.

Methods of engineering TAL to bind to specific nucleic acids aredescribed in Cermak, et al, Nucl. Acids Res. 1-11 (2011). US PublishedApplication No. 2011/0145940, which discloses TAL effectors and methodsof using them to modify DNA. Miller et al. Nature Biotechnol 29: 143(2011) reported making TALENs for site-specific nuclease architecture bylinking TAL truncation variants to the catalytic domain of Foldnuclease. The resulting TALENs were shown to induce gene modification inimmortalized human cells. General design principles for TALE bindingdomains can be found in, for example, WO 2011/072246.

b. Donor Polynucleotides

The nuclease activity of the genome editing systems described hereincleave target DNA to produce single or double strand breaks in thetarget DNA. Double strand breaks can be repaired by the cell in one oftwo ways: non-homologous end joining, and homology-directed repair. Innon-homologous end joining (NHEJ), the double-strand breaks are repairedby direct ligation of the break ends to one another. As such, no newnucleic acid material is inserted into the site, although some nucleicacid material may be lost, resulting in a deletion. In homology-directedrepair (HDR), a donor polynucleotide with homology to the cleaved targetDNA sequence is used as a template for repair of the cleaved target DNAsequence, resulting in the transfer of genetic information from a donorpolynucleotide to the target DNA. As such, new nucleic acid material canbe inserted/copied into the site.

Therefore, in some embodiments, the nucleic acid cargo is or includes adonor polynucleotide. The modifications of the target DNA due to NHEJand/or homology-directed repair can be used to induce gene correction,gene replacement, gene tagging, transgene insertion, nucleotidedeletion, gene disruption, gene mutation, etc.

Accordingly, cleavage of DNA by the genome editing composition can beused to delete nucleic acid material from a target DNA sequence bycleaving the target DNA sequence and allowing the cell to repair thesequence in the absence of an exogenously provided donor polynucleotide.Alternatively, if the genome editing composition includes a donorpolynucleotide sequence that includes at least a segment with homologyto the target DNA sequence, the methods can be used to add, i.e., insertor replace, nucleic acid material to a target DNA sequence (e.g., to“knock in” a nucleic acid that encodes for a protein, an siRNA, anmiRNA, etc.), to add a tag (e.g., 6xHis, a fluorescent protein (e.g., agreen fluorescent protein; a yellow fluorescent protein, etc.),hemagglutinin (HA), FLAG, etc.), to add a regulatory sequence to a gene(e.g., promoter, polyadenylation signal, internal ribosome entrysequence (IRES), 2A peptide, start codon, stop codon, splice signal,localization signal, etc.), to modify a nucleic acid sequence (e.g.,introduce a mutation), and the like. As such, the compositions can beused to modify DNA in a site-specific, i.e., “targeted”, way, forexample gene knock-out, gene knock-in, gene editing, gene tagging, etc.as used in, for example, gene therapy.

In applications in which it is desirable to insert a polynucleotidesequence into a target DNA sequence, a polynucleotide including a donorsequence to be inserted is also provided to the cell. By a “donorsequence” or “donor polynucleotide” or “donor oligonucleotide” it ismeant a nucleic acid sequence to be inserted at the cleavage site. Thedonor polynucleotide typically contains sufficient homology to a genomicsequence at the cleavage site, e.g., 70%, 80%, 85%, 90%, 95%, or 100%homology with the nucleotide sequences flanking the cleavage site, e.g.,within about 50 bases or less of the cleavage site, e.g., within about30 bases, within about 15 bases, within about 10 bases, within about 5bases, or immediately flanking the cleavage site, to supporthomology-directed repair between it and the genomic sequence to which itbears homology. The donor sequence is typically not identical to thegenomic sequence that it replaces. Rather, the donor sequence maycontain at least one or more single base changes, insertions, deletions,inversions or rearrangements with respect to the genomic sequence, solong as sufficient homology is present to support homology-directedrepair. In some embodiments, the donor sequence includes anon-homologous sequence flanked by two regions of homology, such thathomology-directed repair between the target DNA region and the twoflanking sequences results in insertion of the non-homologous sequenceat the target region.

2. Immunomodulation

a. CAR T Cells

The disclosed compositions and methods are particularly useful in thecontext of preparing lymphocytes expressing immune receptors,particularly chimeric immune receptors (CIR) such as chimeric antigenreceptors (CAR). Artificial immune receptors (also known and referred toherein, as chimeric T cell receptors, chimeric immunoreceptors, chimericantigen receptors (CARs), and chimeric immune receptors (CIR)) areengineered receptors, which graft a selected specificity onto a cell.Cells modified according to the discussed methods can be utilized, asdiscussed in more detail below, in a variety of immune therapies fortreatment of cancers, infections, inflammation, and autoimmune diseases.

In particularly preferred embodiments, mRNA or DNA encoding a chimericantigen receptor cargo is delivered to immune cells, such aslymphocytes.

The cargo can be delivered to immune cells in vivo, ex vivo, or invitro. In preferred embodiments, the cargo is mRNA, which may allow forone or more of reduced cost, ease of manufacturing, reduced side effects(e.g., cytokine storm, neurotoxicity, graft vs. host diseases, etc.). Ina particular embodiments, immune cells (e.g., T cells) are harvestedfrom a subject in need of CAR T cell therapy, the compositions andmethods disclosed herein are used to deliver mRNA encoding one or moreCAR T cell constructs into the harvested cells, and the cells arereturned to the subject. In some embodiments, the process, frominitially harvesting the cells to returning them to the subject, takes 1week or less, for example, 1, 2, 3, 4, 5, 6, or 7 days. In particularembodiments, the process, from initially harvesting the cells toreturning them to subject is carried in out in 1 or 2 days, or in lessthan 1 days, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, or 23 hours.

Strategies for the design and development of chimeric antigen receptorsare reviewed in Dotti, et al., Immunol Rev. 2014 January; 257(1):doi:10.1111/imr.12131 (35 pages), which is a specifically incorporatedby reference herein in its entirety, as well as Dotti, MolecularTherapy, 22(5):899-890 (2014), Karlsson, et al., Cancer Gene Therapy,20:386-93 (2013), Charo, et al., Cancer Res., 65(5):2001-8 (2005),Jensen, et al., Immunol Rev., 257(1): 127-144 (2014), Eaton, et al.,Gene Therapy, 9:527-35 (2002), Barrett, et al., Annu Rev Med., 65:333-347 (2014), Cartellieri, et al., Journal of Biomedicine andBiotechnology, Volume 2010, Article ID 956304, 13 pagesdoi:10.1155/2010/956304; and U.S. Published Application Nos.2015/0017120, 2015/0283178, 2015/0290244, 2014/0050709, and2013/0071414.

CARs combine the antigen-binding property of monoclonal antibodies withthe lytic capacity and self-renewal of T cells and have severaladvantages over conventional T cells (Ramos and Dotti, Expert Opin BiolTher., 11:855-873 (2011), Curran, et al., J Gene Med., 14:405-415(2012), Maher, ISRN Oncol. 2012:278093 (2012)). CAR-T cells recognizeand kill cancer cells independently of the major histocompatibilitycomplex (MHC). Thus target cell recognition is unaffected by some of themechanisms by which tumors evade MHC-restricted T-cell recognition, forexample downregulation of human leukocyte antigen (HLA) class Imolecules and defective antigen processing.

Chimeric immune receptors were initially developed in the 1980s andoriginally included the variable (antigen binding) regions of amonoclonal antibody and the constant regions of the T-cell receptor(TCR) a and 13 chains (Kuwana, et al., Biochem Biophys Res Commun.,149:960-968 (1987)). In 1993 this design was modified to include anectodomain, from a single chain variable fragment (scFv) from theantigen binding regions of both heavy and light chains of a monoclonalantibody, a transmembrane domain, and an endodomain with a signalingdomain derived from CD3-ζ. Later CARs have generally followed a similarstructural design, with a co-stimulatory signaling endodomain.Accordingly, the CAR constructs utilized in the methods herein caninclude an antigen binding domain or ectodomain, a hinge domain, atransmembrane domain, an endodomain, and combinations thereof.

In some embodiments the ectodomain is an scFv. The affinity of the scFvpredicts CAR function (Hudecek, et al., Clin Cancer Res., 19(12):3153-64(2013), Chmielewski, et al., J Immunol., 173:7647-7653 (2004)). Antigenbinding and subsequent activation can also be modified by adding aflexible linker sequence in the CAR, which allows for expression of twodistinct scFvs that can recognize two different antigens (Grada, et al.,Mol Ther Nucleic Acids, 2:e105 (2013)) (referred to as tandem CARs(TanCARs)). Tandem CARS may be more effective in killing cancersexpressing low levels of each antigen individually and may also reducethe risk of tumor immune escape due by single antigen loss variants.Other ectodomains include IL13Rα2 (Kahlon, et al., Cancer Res.,64:9160-9166 (2004), Brown, et al., Clin Cancer Res., 18(8):2199-209(2012), Kong, et al., Clin Cancer Res., 18:5949-5960 (2012),NKG2D-ligand and CD70 receptor, peptide ligands (e.g., T1E peptideligand), and so-called “universal ectodomains” (e.g., avidin ectodomaindesigned to recognize targets that have been contacted with biotinylatedmonoclonal antibodies, or FITC-specific scFv designed to recognizetargets that have been contacted with FITC-labeled monoclonal antibodies(Zhang, et al., Blood, 106:1544-1551 (2005), Barber, et al., ExpHematol., 36:1318-1328 (2008), Shaffer, et al., Blood, 117:4304-4314(2011), Davies, et al., Mol Med., 18:565-576 (2012), Urbanska, et al.,Cancer Res., 72:1844-1852 (2012), Tamada, et al., Clin Cancer Res.,18:6436-6445 (2012)).

In some embodiments, the CAR includes a hinge region. While theectodomain is important for CAR specificity, the sequence connecting theectodomain to the transmembrane domain (the hinge region) can alsoinfluence CAR-T-cell function by producing differences in the length andflexibility of the CAR. Hinges can include, for example, a CH2CH3 hinge,or a fragment thereof, derived from an immunoglobulin such as IgG1. Forexample, Hudecek et al. (Hudecek, et al., Clin Cancer Res.,19(12):3153-64 (2013)) compared the influence of a CH2-CH3 hinge [229amino acids (AA)], CH3 hinge (119 AA), and short hinge (12AA) on theeffector function of T cells expressing 3rd generation ROR1-specificCARs and found that T cells expressing ‘short hinge’ CARs had superiorantitumor activity, while other investigators found that a CH2-CH3 hingeimpaired epitope recognition of a 1st generation CD30-specific CAR(Hombach, et al., Gene Ther., 7:1067-1075 (2000)).

Between the hinge (or ectodomain if no hinge domain) and the signalingendodomains typically lies a transmembrane domain, most typicallyderived from CD3-ζ, CD4, CD8, or CD28 molecules. Like hinges, thetransmembrane domain can also influence CAR-T-cell effector function.

Upon antigen recognition, CAR endodomains transmit activation andcostimulatory signals to T cells. T-cell activation relies on thephosphorylation of immunoreceptor tyrosine-based activation motifs(ITAMs) present in the cytoplasmic domain to the cytoplasmic CD3-domainof the TCR complex (Irving, et al., Cell, 64:891-901 (1991)). Althoughthe majority of CAR endomains contain an activation domain derived fromCD3-ζ, others can include ITAM-containing domains such as the Fcreceptor for IgE-γ domain (Haynes, et al., J Immunol., 166:182-187(2001)).

The target specificity of the cell expressing a CAR is determined by theantigen recognized by the antibody/ectodomain. The disclosedcompositions and methods can be used to create constructs, and cellsexpressing the constructs, that target any antigen. In the context ofimmunotherapy, particularly cancer immunotherapy, numerous antigens, andsuitable ectodomains for targeting them, are well known. Unlike thenative TCR, the majority of scFv-based CARs recognize target antigensexpressed on the cell surface rather than internal antigens that areprocessed and presented by the cells' MHC, however, CARs have theadvantage over the classical TCR that they can recognize structuresother than protein epitopes, including carbohydrates and glycolipidsDotti, et al., Immunol Rev. 2014 January; 257(1): doi:10.1111/imr.12131(35 pages) thus increasing the pool of potential target antigens.Preferred targets include antigens that are only expressed on cancercells or their surrounding stroma (Cheever, et al., Clin Cancer Res.,15:5323-5337 (2009)), such as the splice variant of EGFR (EGFRvIII),which is specific to glioma cells (Sampson, et al., Semin Immunol.,20(5):267-75 (2008)). However, human antigens meet this requirement, andthe majority of target antigens are expressed either at low levels onnormal cells (e.g. GD2, CAIX, HER2) and/or in a lineage restrictedfashion (e.g. CD19, CD20).

Preferred targets, and CARs that target them are known in the art (see,e.g., Dotti, et al., Immunol Rev. 2014 January; 257(1):doi:10.1111/imr.12131 (35 pages). For example, CAR targets forhematological malignancies include, but are not limited to, CD 19 (e.g.,B-cell) (Savoldo, et al., J Clin Invest., 121:1822-1826 (2011), Cooper,et al., Blood, 105:1622-1631 (2005); Jensen, et al., Biol Blood MarrowTransplant (2010), Kochenderfer, et al., Blood, 119:2709-2720 (2012),Brentjens, et al., Molecular Therapy, 17:S157 (2009), Brentjens, et al.,Nat Med., 9:279-286 (2003), Brentjens, et al., Blood, 118:4817-4828(2011), Porter, et al., N Engl J Med., 365:725-733 (2011), Kalos, etal., Sci Transl Med., 3:95ra73 (2011), Brentjens, et al., Sci TranslMed., 5:177ra38 (2013), Grupp, et al., N Engl J Med (2013)); CD20 (e.g.,B-cell) (Jensen, et al., Biol Blood Marrow Transplant (2010), Till, etal., Blood, 112:2261-2271 (2008), Wang, et al., Hum Gene Ther.,18:712-725 (2007), Wang, et al., Mol Ther., 9:577-586 (2004), Jensen, etal., Biol Blood Marrow Transplant, 4:75-83 (1998)); CD22 (e.g., B-cell)(Haso, et al., Blood, 121:1165-1174 (2013)); CD30 (e.g., B-cell) (DiStasi, et al., Blood, 113:6392-6402 (2009), Savoldo, et al., Blood,110:2620-2630 (2007), Hombach, et al., Cancer Res., 58:1116-1119(1998)); CD33 (e.g., Myeloid) (Finney, et al., J Immunol., 161:2791-2797(1998)); CD70 (e.g., B-cell/T-cell) (Shaffer, et al., Blood,117:4304-4314 (2011)); CD123 (e.g., Myeloid) (Tettamanti, et al., Br JHaematol., 161:389-401 (2013)); Kappa (e.g., B-cell) (Vera, et al.,Blood, 108:3890-3897 (2006)); Lewis Y (e.g., Myeloid) (Peinert, et al.,Gene Ther., 17:678-686 (2010), Ritchie, et al., Mol Ther. (2013)); NKG2Dligands (e.g., Myeloid) (Barber, et al., Exp Hematol., 36:1318-1328(2008), Lehner, et al., PLoS One., 7:e31210 (2012), Song, et al., HumGene Ther., 24:295-305 (2013), Spear, et al., J Immunol. 188:6389-6398(2012)); ROR1 (e.g., B-cell) (Hudecek, et al., Clin Cancer Res. (2013)).

CAR targets for solid tumors include, but are not limited to, B7H3(e.g., sarcoma, glioma) (Cheung, et al., Hybrid Hybridomics, 22:209-218(2003)); CAIX (e.g., kidney) (Lamers, et al., J Clin Oncol., 24:e20-e22.(2006)), Weijtens, et al., Int J Cancer, 77:181-187 (1998)); CD44 v6/v7(e.g., cervical) (Hekele, et al., Int J Cancer, 68:232-238 (1996)),Dall, et al., Cancer Immunol Immunother, 54:51-60 (2005); CD171 (e.g.,neuroblastoma) (Park, et al., Mol Ther., 15:825-833 (2007)); CEA (e.g.,colon) (Nolan, et al., Clin Cancer Res., 5:3928-3941 (1999)); EGFRvIII(e.g., glioma) (Bullain, et al., J NeurooncoL (2009), Morgan, et al.,Hum Gene Ther., 23:1043-1053 (2012)); EGP2 (e.g., carcinomas) (Meier, etal., Magn Reson Med., 65:756-763 (2011), Ren-Heidenreich, et al., CancerImmunol Immunother., 51:417-423 (2002)); EGP40 (e.g., colon) (Daly, etal., Cancer Gene Ther., 7:284-291 (2000); EphA2 (e.g., glioma, lung)(Chow, et al., Mol Ther., 21:629-637 (2013)); ErbB2 (HER2) (e.g.,breast, lung, prostate, glioma) (Zhao, et al., J Immunol., 183:5563-5574(2009), Morgan, et al., Mol Ther., 18:843-851 (2010), Pinthus, et al.,114:1774-1781 (2004), Teng, et al., Hum Gene Ther., 15:699-708 (2004),Stancovski, et al., J Immunol., 151:6577-6582 (1993), Ahmed, et al., MolTher., 17:1779-1787 (2009), Ahmed, et al., Clin Cancer Res., 16:474-485(2010), Moritz, et al., Proc Natl Acad Sci U.S.A., 91:4318-4322 (1994));ErbB receptor family (e.g., breast, lung, prostate, glioma) (Davies, etal., Mol Med., 18:565-576 (2012)); ErbB3/4 (e.g., breast, ovarian)(Muniappan, et al., Cancer Gene Ther., 7:128-134 (2000), Altenschmidt,et al., Clin Cancer Res., 2:1001-1008 (1996)); HLA-A1/MAGE1 (e.g.,melanoma) (Willemsen, et al., Gene Ther., 8:1601-1608 (2001), Willemsen,et al., J Immunol., 174:7853-7858 (2005)); HLA-A2/NY-ESO-1 (e.g.,sarcoma, melanoma) (Schuberth, et al., Gene Ther., 20:386-395 (2013));FR-α (e.g., ovarian) (Hwu, et al., J Exp Med., 178:361-366 (1993),Kershaw, et al., Nat Biotechnol., 20:1221-1227 (2002), Kershaw, et al.,Clin Cancer Res., 12:6106-6115 (2006), Hwu, et al., Cancer Res.,55:3369-3373 (1995)); FAP (e.g., cancer associated fibroblasts)(Kakarla, et al., Mol Ther. (2013)); FAR (e.g., rhabdomyosarcoma)(Gattenlohner, et al., Cancer Res., 66:24-28 (2006)); GD2 (e.g.,neuroblastoma, sarcoma, melanoma) (Pule, et al., Nat Med., 14:1264-1270(2008), Louis, et al., Blood, 118:6050-6056 (2011), Rossig, et al., IntJ Cancer., 94:228-236 (2001)); GD3 (e.g., melanoma, lung cancer) (Yun,et al., Neoplasia., 2:449-459 (2000)); HMW-MAA (e.g., melanoma) (Burns,et al., Cancer Res., 70:3027-3033 (2010)); IL11Rα (e.g., osteosarcoma)(Huang, et al., Cancer Res., 72:271-281 (2012)); IL13Rα2 (e.g., glioma)(Kahlon, et al., Cancer Res., 64:9160-9166 (2004), Brown, et al., ClinCancer Res. (2012), Kong, et al., Clin Cancer Res., 18:5949-5960 (2012),Yaghoubi, et al., Nat Clin Pract Oncol., 6:53-58 (2009)); Lewis Y (e.g.,breast/ovarian/pancreatic) (Peinert, et al., Gene Ther., 17:678-686(2010), Westwood, et al., Proc Natl Acad Sci U.S.A., 102:19051-19056(2005), Mezzanzanica, et al., Cancer Gene Ther., 5:401-407 (1998));Mesothelin (e.g., mesothelioma, breast, pancreas) (Lanitis, et al., MolTher., 20:633-643 (2012), Moon, et al., Clin Cancer Res., 17:4719-4730(2011)); Muel (e.g., ovarian, breast, prostate) (Wilkie, et al., JImmunol., 180:4901-4909 (2008)); NCAM (e.g., neuroblastoma, colorectal)(Gilham, et al., J Immunother., 25:139-151 (2002)); NKG2D ligands (e.g.,ovarian, sacoma) (Barber, et al., Exp Hematol., 36:1318-1328 (2008),Lehner, et al., PLoS One, 7:e31210 (2012), Song, et al., Gene Ther.,24:295-305 (2013), Spear, et al., J Immunol., 188:6389-6398 (2012));PSCA (e.g., prostate, pancreatic) (Morgenroth, et al., Prostate,67:1121-1131 (2007), Katari, et al., HPB, 13:643-650 (2011)); PSMA(e.g., prostate) (Maher, et al., Nat BiotechnoL, 20:70-75 (2002), Gong,et al., Neoplasia., 1:123-127 (1999)); TAG72 (e.g., colon) (Hombach, etal., Gastroenterology, 113:1163-1170 (1997), McGuinness, et al., HumGene Ther., 10:165-173 (1999)); VEGFR-2 (e.g., tumor vasculature) (JClin Invest., 120:3953-3968 (2010), Niederman, et al., Proc Natl AcadSci U.S.A., 99:7009-7014 (2002)).

b. Metabolic Stability

In some embodiments, cells' (e.g., CAR cells') metabolic stability isimproved by equipping them with the capacity to make the very growthfactors that are limiting in vivo. In some embodiments, nucleic acidcargo encoding an anti-apoptotic factor such as BCL-XL is transientlydelivered to cells. B-cell lymphoma-extra large (Bcl-XL, or BCL2-like 1isoform 1) is a transmembrane protein in the mitochondria. It is amember of the Bcl-2 family of proteins, and acts as a pro-survivalprotein in the intrinsic apoptotic pathway by preventing the release ofmitochondrial contents such as cytochrome c, which would lead to caspaseactivation. Both amino acid and nucleic acid sequences encoding BCL-XLare known in the art and include, for example, UniProtKB-Q07817(B2CL1_HUMAN), Isoform Bcl-X(L) (identifier: Q07817-1) (amino acidsequence); ENA|U72398|U72398.1 Human Bcl-x beta (bcl-x) gene, completecds (genomic nucleic acid sequences); ENA|Z23115|Z23115.1 H.sapiensbcl-XL mRNA (mRNA/cDNA nucleic acid sequences).

In some embodiments, the nucleic cargo encodes a proliferation inducingfactor such as IL-2. Both amino acid and nucleic acid sequences encodingIL-2 are known in the art and include, for example, UniProtKB-P60568(IL2_HUMAN) (amino acid sequence); ENA|X00695|X00695.1 Humaninterleukin-2 (IL-2) gene and 5′ flanking region (genic nucleic acidsequence); and ENA|V00564|V00564.1 Human mRNA encoding interleukin-2(IL-2) (mRNA/cDNA nucleic acid sequence).

However, the production of secreted IL-2 may have the unwanted sideeffect of also stimulating the proliferation of the lymphoma and Tregcells, and impairing the formation of memory T cells (Zhang, et al.,Nature Medicine, 11:1238-1243 (2005)). In addition, the use of IL-2 inpatients treated with Tumor Infiltrating Lymphocytes (TILs) led toincreased toxicity (Heemskerk, et al., Human Gene Therapy, 19:496-510(2008)). To avoid this potentiality, in addition or alternative to IL-2,the nucleic acid cargo can encode a chimeric γc cytokine receptor (CγCR)such as one composed of Interleukin-7 (IL-7) tethered to IL-7Rα/CD127that confers exogenous cytokine independent, cell intrinsic, STAT5cytokine signals (Hunter, et al., Molecular Immunology, 56:1-11 (2013)).The design is modular in that the IL-2Rβ/CD122 cytoplasmic chain can beexchanged for that of IL-7Rα/CD127, to enhance Shc activity. Theconstructs mimic wild type IL-2 signaling in human CD8+ T cells (Hunter,et al., Molecular Immunology, 56:1-11 (2013)) and should, therefore,work similarly to the IL-2 mRNA, without the unwanted to side effects.

Additionally and alternatively other antiapoptotic molecules andcytokines can be used to preserve cell viability in the native state.Exemplary factors include, but are not limited to:

Myeloid Cell Leukemia 1 (MCL-1) (e.g., UniProtKB-Q07820 (MCL1_HUMAN)(amino acid sequence); ENA|AF147742|AF147742.1 Homo sapiens myeloid celldifferentiation protein (MCL1) gene, promoter and complete cds (genomicnucleic acid sequence); ENA|AF118124|AF118124.1 Homo sapiens myeloidcell leukemia sequence 1 (MCL1) mRNA, complete cds. (mRNA/cDNA nucleicacid sequence)) which is an antiapoptotic factor;

IL-7 (e.g., UniProtKB-P13232 (IL7_HUMAN) (amino acid sequence);ENA|EF064721|EF064721.1 Homo sapiens interleukin 7 (IL7) gene, completecds. (genomic nucleic acid sequence); ENA|J04156|J04156.1 Humaninterleukin 7 (IL-7) mRNA, complete cds. (mRNA/cDNA nucleic acidsequence) which is important for T cell survival and development, and

IL-15 (e.g., UniProtKB-P40933 (IL15_HUMAN) (amino acid sequence);ENA|X91233|X91233.1 H.sapiens IL15 gene (genomic nucleic acid sequence);ENA|U14407|U14407.1 Human interleukin 15 (IL15) mRNA, complete cds.(mRNA/cDNA nucleic acid sequence)) which promotes T and NK cell survival(Opferman, et al., Nature, 426: 671-676 (2003); Meazza, et al., Journalof Biomedicine & Biotechnology, 861920, doi:10.1155/2011/861920 (2011);Michaud, et al., Journal of Immunotherapy, 33:382-390 (2010)). Thesecytokine mRNAs can be used either independently or in combination withBCL-XL, IL-2, and/or CγCR mRNA. Accordingly, in some embodiments, anmRNA encoding MCL-1, IL-7, IL-15, or a combination thereof is deliveredto cells.

c. Inhibitory CAR (iCAR)

In some embodiments, T cell therapies are delivered to the CAR cellsthat have demonstrated long-term efficacy and curative potential for thetreatment of some cancers, however, their use is limited by damage tonon-cancerous tissues reminiscent of graft-versus-host disease afterdonor lymphocyte infusion. Any of the disclosed compositions and methodscan be used in combination with a non-specific immunosuppression (e.g.,high-dose corticosteroid therapy, which exert cytostatic or cytotoxiceffects on T cells, to restrain immune responses), irreversible T cellelimination (e.g., so-called suicide gene engineering strategies), or acombination thereof. However, in some preferred embodiments, off-targeteffects are reduced by introducing into the CAR cell a constructencoding an inhibitory chimeric antigen receptor (iCAR). T cells withspecificity for both tumor and off-target tissues can be restricted totumor only by using an antigen-specific iCAR introduced into the T cellsto protect the off-target tissue (Fedorov, et al., Science TranslationalMedicine, 5:215ra172 (2013)). The iCAR can include a surface antigenrecognition domain combined with a powerful acute inhibitory signalingdomain to limit T cell responsiveness despite concurrent engagement ofan activating receptor (e.g., a CAR). In preferred embodiments, the iCARincludes a single-chain variable fragment (scFv) specific for aninhibitory antigen fused to the signaling domains of an immunoinhibitoryreceptor (e.g., CTLA-4, PD-1, LAG-3, 2B4 (CD244), BTLA (CD272), KIR,TIM-3, TGF beta receptor dominant negative analog etc.) via atransmembrane region that inhibits T cell function specifically uponantigen recognition. Once the CAR cell encounters a cell (e.g., a cancercell) that does not express the inhibitory antigen, iCAR-transduced Tcells can mount a CAR-induced response against the CAR's target antigen.A DNA iCAR using an scFv specific for PSMA with the inhibitory signalingdomains of either CTLA-4 or PD-1 is discussed in (Fedorov, et al.,Science Translational Medicine, 5:215ra172 (2013)).

Design considerations include that observation that PD-1 was a strongerinhibitor than CTLA-4, CTLA-4 exhibited cytoplasmic localization unlessa Y165G mutant was used, and that the iCAR expression level isimportant.

iCAR can be designed against cell type specific surface molecules. Insome embodiments the iCAR is designed to prevent T cells, NK cells, orother immune cell reactivity against certain tissues or cell types.

d. Reducing Endogenous Inhibitory Signaling

In some embodiments the cells are contacted with a nucleic acid cargothat reprograms the cells to prevent expression of one or more antigens.For example, in some embodiments the nucleic acid cargo is or encodes aninterfering RNA that prevents expression of an mRNA encoding antigenssuch as CTLA-4 or PD-1. This method can be used to prepare universaldonor cells. RNAs used to alter the expression of allogenic antigens maybe used alone or in combination with RNAs that result inde-differentiation of the target cell.

Although the section above provides compositions and methods thatutilized inhibitory signaling domains e.g., from CTLA-4 or PD-1 in anartificial iCAR to restrict on-target/off-tumor cytotoxicity,additionally or alternatively overall CAR cell on-tumor effectorefficiency can be increased by reducing the expression of endogenousinhibitory signaling in the CAR cells so that the CAR cells becomeresistant to the inhibitory signals of the hostile tumormicroenvironment.

CTLA-4 and PD-1 inhibit T cells at different stages in activation andfunction. CTLA-4 regulates T cell responses to self-antigens, asknockout mice spontaneously develop organ damage due to highly active,tissue-infiltrating T cells without specific antigen exposure (Tivol, etal., Immunity, 3:541-547 (1995); Waterhouse, et al., Science,270:985-988 (1995)). Interestingly, conditional knockout of CTLA-4 inTreg cells recapitulates the global knockout indicating that it normallyfunctions within Tregs (Wing, et al., Science, 322:271-275 (2008)). Incontrast, PD-L1 knockout mice are autoimmune prone, but do notspontaneously develop massive inflammatory cell infiltration of normalorgans, indicating that it's major physiological function is to mediatenegative feedback control of ongoing tissue inflammation in an induciblemanner (Dong, et al., Immunity, 20:327-336 (2004)). Indeed, according tothe “adaptive resistance” hypothesis most tumors up-regulate PD-L1 inresponse to IFNγ; a key cytokine released by effector T cells includingCART cells (Greenwald, et al., Annu Rev Immunol, 23:515-548 (2005);Carreno, et al., Annu Rev Immunol, 20:29-53 (2002); Chen, et al., TheJournal of Clinical Investigation, 125:3384-3391 (2015); Keir, et al.,Annu Rev Immunol, 26:677-704 (2008); Pentcheva-Hoang, et al.,Immunological Reviews, 229:67-87 (2009)). PD-L1 then delivers aninhibitory signal to T cells decreasing their proliferation, andcytokine and perforin production (Butte, et al., Immunity, 27:111-122(2007); Chen, et al., Immunology, 4:336-347 (2004); Park, et al., Blood,116:1291-1298 (2010); Wherry, et al., Nat Immunol, 12:492-499 (2011);Zou, et al., Immunology, 8:467-477 (2008)). In addition, reversesignaling from the T cell through B7-H1 on cancer cells induces ananti-apoptotic effect that counteracts Fas-L signaling (Azuma, et al.,Blood, 111:3635-3643 (2008)). Azuma, et al., Blood, 111:3635-3643 (2008)

In light of the up-regulation of B7-H1 by cancer cells and theassociation of its expression with cancer progression and poor clinicaloutcome (Flies, et al., Journal of Immunotherapy, 30:251-260 (2007);Nishimura, et al., Immunity, 11:141-151 (1999); Wang, et al., Curr TopMicrobiol Immunol, 344:245-267 (2011)), antibodies antagonizing the PD-1and CTLA-4 pathways have shown dramatic efficacy in solid tumors,particularly melanoma, with the combination of the two showing even moreactivity. The anti-CTLA-4 antibody, ipilimumab, improves overallsurvival in metastatic melanoma with increased T cell infiltration intotumors and increased intratumoral CD8+:Treg ratios, predominantlythrough inhibition of Treg cells (Hamid, et al., J Transl Med, 9:204(2011); Ribas, et al., Clinical Cancer Research: An Official Journal ofthe American Association for Cancer Research, 15:6267-6276 (2009);Twyman-Saint, et al., Nature, 520:373-377 (2015)). The anti-PD-1antibody, nivolumab, shows an overall response rate of 30-40% inmetastatic melanoma (Robert, et al., The New England Journal ofMedicine, 372:320-330 (2015); Topalian, et al., J Clin Oncol,32:1020-1030 (2014)), with similar findings in early phase clinicaltrials for other solid tumors including metastatic renal cancer,non-small cell lung cancer and relapsed Hodgkin's Lymphoma (Ansell, etal., The New England Journal of Medicine, 372:311-319 (2015); Brahmer,et al., J Clin Oncol, 28:3167-3175 (2010); Topalian, et al., The NewEngland Journal of Medicine, 366:2443-2454 (2012)). As resistance toanti-CTLA-4 antibodies in mouse melanoma models is due to up-regulationof PD-L181, the combination of both ipilimumab and nivolumabdemonstrates further efficacy in both mouse models and human patients(Larkin, et al., The New England Journal of Medicine, 373:23-34 (2015);Spranger, et al., J Immunother Cancer, 2, 3, doi:10.1186/2051-1426-2-3(2014); Yu, et al., Clinical Cancer Research: An Official Journal of theAmerican Association for Cancer Research, 16:6019-6028 (2010)). Giventhe importance of the checkpoint inhibition pathway, it is believed thatPD-1/CTLA-4 inhibition will release the brake, while the chimericantigen receptor will push on the gas pedal. Importantly, transientdelivery can be utilized to only transiently release the brake so thatthese cells will not lead to future autoimmune disease.

i. CRISPRi

To avoid permanent genome modification and inactivation of inhibitorysignals such as PD-1 and CTLA-4, the dCAS9 CRISPRi system (Larson, etal., Nat Protoc, 8:2180-2196 (2013)) can be utilized. Nucleic acidsencoding the enzymatically-inactive dCAS9-KRAB-repression domain, fusionprotein, and sgRNAs to the inhibitory signaling protein (e.g. CTLA-4,PD-1, LAG-3, 2B4 (CD244), BTLA (CD272), KIR, TIM-3, TGF beta receptordominant negative analog, etc.) can be co-delivered into the CAR cell.One or multiple sgRNA can be utilized. sgRNA can be designed to targetthe proximal promoter region and the coding region (nontemplate strand).An alternative approach utilizes the single-component Cpf1 CRISPRsystem, which is a smaller RNA to electroporate and express (Zetsche, etal., Cell, doi:10.1016/j.ce11.2015.09.038 (2015)). Any of the foregoingRNA components can also be encoded by DNA expression construct such as avector, for example a plasmid. Thus, either RNA, DNA, or a combinationthereof can serve as the nucleic acid cargo.

Although broad inhibition of CTLA-4 with ipilimumab results inautoimmune sequelae, it is believed these side-effects will be decreasedby restricting loss to CAR cells and transient nature of the mRNAdelivery. Inhibitory function will be regained in time.

ii. Inhibitory RNAs

Nucleic acid cargo that can be delivered to cells can be or encode afunctional nucleic acid or polypeptide designed to target and reduce orinhibit expression or translation of an inhibitory signaling moleculemRNA; or to reduce or inhibit expression, reduce activity, or increasedegradation of inhibitory signaling molecule protein. Suitabletechnologies include, but are not limited to, antisense molecules,siRNA, miRNA, aptamers, ribozymes, triplex forming molecules, RNAi, etc.In some embodiments, the mRNA encode antagonist polypeptide that reduceinhibitory signaling.

In some embodiments, cargo that is or encodes functional RNAs suitableto reducing or silencing expression of CTLA-4, PD-1, LAG-3, 2B4 (CD244),BTLA (CD272), KIR, TIM-3, TGF beta receptor dominant negative analog,etc. alone or in combination can be delivered to cells.

In some embodiments, the cargo is an RNA or DNA that encodes apolypeptide that reduces bioavailability or serves as an antagonist orother negative regulator or inhibitor of CTLA-4, PD-1, LAG-3, 2B4(CD244), BTLA (CD272), KIR, TIM-3, TGF beta receptor dominant negativeanalog, or another protein in an immune inhibitory pathway. The proteincan be a paracrine, endocrine, or autocrine. It can regulate the cellintracellularly. It can be secreted and regulate the expressing celland/or other (e.g., neighboring) cells. It can be a transmembraneprotein that regulates the expressing cell and/or other cells. Theprotein can be fusion protein, for example an Ig fusion protein.

e. Pro-apoptotic Factors

Compositions and methods for activating and reactivating apoptoticpathways are also provided. In some embodiments, the nucleic acid is orencodes a factor or agent that activates, reactivates, or otherwiseenhances or increases the intrinsic apoptosis pathway. Preferably thefactor activates, reactivates, or otherwise enhances the intrinsicapoptosis pathway in cancer (e.g., tumor) cells, and is more preferablyspecific or targeted to the cancer cells.

In some embodiments, cells, following delivery of an anti-apoptoticfactor or pro-proliferation factor, such as those discussed above orotherwise known in the art, are more resistant or less sensitive toinduced apoptosis than untreated cells. A pro-apoptotic factor caninduce or increase apoptosis in, for example, untreated cells relativeto the treated T cells, and is preferably selective for cancer cells.The regimen results in a two-pronged attack, one cellular and onemolecular, against the cancer cells.

The intrinsic apoptosis pathway can be activated, reactivated, orotherwise enhanced by targeting BCL-2 family members. BCL-2 familymembers are classified into three subgroups based on function and Bcl-2Homology (BH) domains: multi-domain anti-apoptotic (e.g. BCL-2 orBCL-XL), multi-domain pro-apoptotic (e.g. BAX and BAK), and BH3-onlypro-apoptotic (e.g. BIM) proteins. Members of the BH3-only subgroup,such as BIM, function as death sentinels that are situated throughoutthe cell, poised to transmit a variety of physiological and pathologicsignals of cellular injury to the core apoptotic machinery located atthe mitochondrion (Danial, et al., Cell, 116:205-219 (2004)).

In some embodiments, the pro-apoptotic factor is a pro-apoptoticBH3-mimetic. Various pro-apoptotic BH3-mimetics can simulate the nativepro-apoptotic activities of BIM and afford the ability to manipulatemultiple points of the apoptotic pathway. For example, BIM SAHB(Stabilized Alpha Helix of BCL-2 domains), ABT-737, and ABT-199 arepro-apoptotic BH3-mimetics designed by structural studies of theinteraction between the pro-apoptotic BH3-only helical domain and thehydrophobic groove formed by the confluence of the BH1, BH2 and BH3domains of anti-apoptotic proteins (Oltersdorf, et al., Nature,435:677-681 (2005)).

D. Target Cells

In some embodiments, one or more particular cell types or tissue is thetarget of the disclosed complexes. The target cells can be in vitro, exvivo or in a subject (i.e., in vivo). The application discussed hereincan be carried out in vitro, ex vivo, or in vivo. For ex vivoapplication, the cells can be collected or isolated and treated inculture. Ex vivo treated cells can be administered to a subject in needthereof in therapeutically effective amount. For in vivo applications,cargo can be delivered to target cells passively, e.g., based oncirculation of the composition, local delivery, etc., or can be activelytargeted, e.g., with the additional a cell, tissue, organ specifictargeting moiety. Thus, in some embodiments, cargo is delivered to thetarget cells to the exclusion of other cells. In some embodiments, cargois delivered to target cells and non-target cells.

Target cells can be selected by the practitioner based on the desiredtreatment and therapy, and the intended effect of the nucleic acidcargo. For example, when the nucleic acid cargo is intended to inducecell death, the target cells may be cancer cells; when the nucleic acidcargo is intended to induce a genomic alteration, the target cells maybe stem cells; when the nucleic acid cargo encodes a chimeric antigenreceptor, the target cells may be immune cells.

3E10 scFv has previously been shown capable of penetrating into cellnuclei in an ENT2-dependent manner, with efficiency of nuclear uptakegreatly impaired in ENT2-deficient cells (Hansen et al., J Biol Chem282, 20790-20793 (2007)). ENT2 (SLC29A2) is a sodium-independenttransporter that participates in the transport of purine and pyrimidinenucleosides and nucleohases, and is less sensitive tonitrobenzylmercaptopurine riboside (NBMPR) than ENT1.

In some embodiments, the target cells express ENT2 on their plasmamember, their nuclear membrane, or both. Expression of ENT2 isrelatively ubiquitous but varies in abundance among tissues and celltypes. It has been confirmed in the brain, heart, placenta, thymus,pancreas, prostate and kidney (Griffiths, et al., Biochem J, 1997. 328(Pt 3): p. 739-43, Crawford, et al., J Biol Chem, 1998. 273 (9): p.5288-93). Relative to other transporters, ENT2 has one of the highestmRNA expressions in skeletal muscle (Baldwin, et al., Pflugers Arch,2004. 447 (5): p. 735-43, Govindarajan, et al., Am J Physiol RegulIntegr Comp Physiol, 2007. 293 (5): p. R1809-22). Thus, in someembodiments the target cells are brain, heart, placenta, thymus,pancreas, prostate, kidney, or skeletal muscle. Due to the highexpression of ENT2 by skeletal muscle, the disclosed compositions andmethods may be particularly effective for delivering nucleic acid cargoto these cells, and/or higher levels of cargo may be delivered to thesecells compared to other cells expressing lower levels of ENT2.

Additional, non-limiting, exemplary target cells are discussed below.

1. Progenitor and Stem Cells

The cells can be hematopoietic progenitor or stem cells. In someembodiments, particularly those related to gene editing and gene therapythe target cells are CD34⁺ hematopoietic stem cells. Hematopoietic stemcells (HSCs), such as CD34+ cells are multipotent stem cells that giverise to all the blood cell types including erythrocytes.

Stem cells can be isolated and enriched by one of skill in the art.Methods for such isolation and enrichment of CD34⁺ and other cells areknown in the art and disclosed for example in U.S. Pat. Nos. 4,965,204;4,714,680; 5,061,620; 5,643,741; 5,677,136; 5,716,827; 5,750,397 and5,759,793. As used herein in the context of compositions enriched inhematopoietic progenitor and stem cells, “enriched” indicates aproportion of a desirable element (e.g. hematopoietic progenitor andstem cells) which is higher than that found in the natural source of thecells. A composition of cells may be enriched over a natural source ofthe cells by at least one order of magnitude, preferably two or threeorders, and more preferably 10, 100, 200 or 1000 orders of magnitude.

In humans, CD34⁺ cells can be recovered from cord blood, bone marrow orfrom blood after cytokine mobilization effected by injecting the donorwith hematopoietic growth factors such as granulocyte colony stimulatingfactor (G-CSF), granulocyte-monocyte colony stimulating factor (GM-CSF),stem cell factor (SCF) subcutaneously or intravenously in amountssufficient to cause movement of hematopoietic stem cells from the bonemarrow space into the peripheral circulation. Initially, bone marrowcells may be obtained from any suitable source of bone marrow, e.g.tibiae, femora, spine, and other bone cavities. For isolation of bonemarrow, an appropriate solution may be used to flush the bone, whichsolution will be a balanced salt solution, conveniently supplementedwith fetal calf serum or other naturally occurring factors, inconjunction with an acceptable buffer at low concentration, generallyfrom about 5 to 25 mM. Convenient buffers include Hepes, phosphatebuffers, lactate buffers, etc.

Cells can be selected by positive and negative selection techniques.Cells can be selected using commercially available antibodies which bindto hematopoietic progenitor or stem cell surface antigens, e.g. CD34,using methods known to those of skill in the art. For example, theantibodies may be conjugated to magnetic beads and immunogenicprocedures utilized to recover the desired cell type. Other techniquesinvolve the use of fluorescence activated cell sorting (FACS). The CD34antigen, which is found on progenitor cells within the hematopoieticsystem of non-leukemic individuals, is expressed on a population ofcells recognized by the monoclonal antibody My-10 (i.e., express theCD34 antigen) and can be used to isolate stem cell for bone marrowtransplantation. My-10 deposited with the American Type CultureCollection (Rockville, Md.) as HB-8483 is commercially available asanti-HPCA 1. Additionally, negative selection of differentiated and“dedicated” cells from human bone marrow can be utilized, to selectagainst substantially any desired cell marker. For example, progenitoror stem cells, most preferably CD34⁺ cells, can be characterized asbeing any of CD3⁻, CD7⁻, CD8⁻, CD10⁻, CD14⁻, CD15⁻, CD19⁻, CD20⁻, CD33⁻,Class II HLA⁺ and Thy-1⁺.

Once progenitor or stem cells have been isolated, they may be propagatedby growing in any suitable medium. For example, progenitor or stem cellscan be grown in conditioned medium from stromal cells, such as thosethat can be obtained from bone marrow or liver associated with thesecretion of factors, or in medium including cell surface factorssupporting the proliferation of stem cells. Stromal cells may be freedof hematopoietic cells employing appropriate monoclonal antibodies forremoval of the undesired cells.

The isolated cells are contacted ex vivo with antibody and nucleic acidcargo complexes. Cells to which cargo has been delivered can be referredto as modified cells. A solution of the complexes may simply be added tothe cells in culture. It may be desirable to synchronize the cells inS-phase. Methods for synchronizing cultured cells, for example, bydouble thymidine block, are known in the art (Zielke, et al., MethodsCell Biol., 8:107-121 (1974)).

The modified cells can be maintained or expanded in culture prior toadministration to a subject. Culture conditions are generally known inthe art depending on the cell type. Conditions for the maintenance ofCD34⁺ in particular have been well studied, and several suitable methodsare available. A common approach to ex vivo multi-potentialhematopoietic cell expansion is to culture purified progenitor or stemcells in the presence of early-acting cytokines such as interleukin-3.It has also been shown that inclusion, in a nutritive medium formaintaining hematopoietic progenitor cells ex vivo, of a combination ofthrombopoietin (TPO), stem cell factor (SCF), and flt3 ligand (Flt-3L;i.e., the ligand of the flt3 gene product) was useful for expandingprimitive (i.e., relatively non-differentiated) human hematopoieticprogenitor cells in vitro, and that those cells were capable ofengraftment in SCID-hu mice (Luens et al., 1998, Blood 91:1206-1215). Inother known methods, cells can be maintained ex vivo in a nutritivemedium (e.g., for minutes, hours, or 3, 6, 9, 13, or more days)including murine prolactin-like protein E (mPLP-E) or murineprolactin-like protein F (mPIP-F; collectively mPLP-E/IF) (U.S. Pat. No.6,261,841). It will be appreciated that other suitable cell culture andexpansion methods can be used as well. Cells can also be grown inserum-free medium, as described in U.S. Pat. No. 5,945,337.

In another embodiment, the modified hematopoietic stem cells aredifferentiated ex vivo into CD4⁺ cells culture using specificcombinations of interleukins and growth factors prior to administrationto a subject using methods well known in the art. The cells may beexpanded ex vivo in large numbers, preferably at least a 5-fold, morepreferably at least a 10-fold and even more preferably at least a20-fold expansion of cells compared to the original population ofisolated hematopoietic stem cells.

In another embodiment cells, can be dedifferentiated somatic cells.Somatic cells can be reprogrammed to become pluripotent stem-like cellsthat can be induced to become hematopoietic progenitor cells. Thehematopoietic progenitor cells can then be treated with the compositionsas described above with respect to CD34⁺ cells. Representative somaticcells that can be reprogrammed include, but are not limited tofibroblasts, adipocytes, and muscles cells. Hematopoietic progenitorcells from induced stem-like cells have been successfully developed inthe mouse (Hanna, J. et al. Science, 318:1920-1923 (2007)).

To produce hematopoietic progenitor cells from induced stem-like cells,somatic cells are harvested from a host. In a preferred embodiment, thesomatic cells are autologous fibroblasts. The cells are cultured andtransduced with vectors encoding Oct4, Sox2, Klf4, and c-Myctranscription factors. The transduced cells are cultured and screenedfor embryonic stem cell (ES) morphology and ES cell markers including,but not limited to AP, SSEA1, and Nanog. The transduced ES cells arecultured and induced to produce induced stem-like cells. Cells are thenscreened for CD41 and c-kit markers (early hematopoietic progenitormarkers) as well as markers for myeloid and erythroid differentiation.

The modified hematopoietic stem cells or modified cells including, e.g.,induced hematopoietic progenitor cells, are then introduced into asubject. Delivery of the cells may be affected using various methods andincludes most preferably intravenous administration by infusion as wellas direct depot injection into periosteal, bone marrow and/orsubcutaneous sites.

The subject receiving the modified cells may be treated for bone marrowconditioning to enhance engraftment of the cells. The recipient may betreated to enhance engraftment, using a radiation or chemotherapeutictreatment prior to the administration of the cells. Upon administration,the cells will generally require a period of time to engraft. Achievingsignificant engraftment of hematopoietic stem or progenitor cellstypically takes weeks to months.

A high percentage of engraftment of modified hematopoietic stem cellsmay not be necessary to achieve significant prophylactic or therapeuticeffect. It is believed that the engrafted cells will expand over timefollowing engraftment to increase the percentage of modified cells. Itis believed that in some cases, engraftment of only a small number orsmall percentage of modified hematopoietic stem cells will be requiredto provide a prophylactic or therapeutic effect.

In preferred embodiments, the cells to be administered to a subject willbe autologous, e.g. derived from the subject, or syngenic.

2. Embryos

In some embodiments, the compositions and methods can be used to delivercargo to embryonic cells in vitro. The methods typically includecontacting an embryo in vitro with an effective amount of antibody-cargoDNA to improve cargo transduction into the embryo. The embryo can be asingle cell zygote, however, treatment of male and female gametes priorto and during fertilization, and embryos having 2, 4, 8, or 16 cells andincluding not only zygotes, but also morulas and blastocytes, are alsoprovided. In some embodiments, the embryo is contacted with thecompositions on culture days 0-6 during or following in vitrofertilization.

The contacting can be adding the compositions to liquid media bathingthe embryo. For example, the compositions can be pipetted directly intothe embryo culture media, whereupon they are taken up by the embryo.

3. Immune Cells

In some embodiments, the target cells are one or more types of immunecells. For example, different type of cells can be utilized or otherwisetargeted for immunodulation and CAR-based therapies. The preferredtargeted/engineered T cells may vary depending on the tumor and goals ofthe adoptive therapy. Effector T cells are typically preferred becausethey secreted high levels of effector cytokines and were proficientkillers of tumor targets in vitro (Barrett, et al., Annu Rev Med., 65:333-347 (2014). Two complimentary lymphocyte populations with robust CARmediated cytotoxicity are CD3-CD56+NK cells and CD3+CD8+ T cells. Use ofCD8+ T cells with CD4+ helper T cells leads to the increased presence ofsuppressive T-reg cells and dampened CD8+ T cell cytotoxicity. Sincereprogrammed CD8+ T cells are pre-activated so that they act directly ontumor cells without the need for activation in the lymph node, CD4+ Tcell support is not essential.

Additionally, there is evidence that infusion of naive T cells(Rosenberg, et al. Adv. Cancer Res., 25:323-388 (1977)), central memoryT cells (T_(CM) cells) (Berger, et al. J. Clin. Invest., 118:294-305(2008)), Th17 cells (Paulos, et al., Sci. Transl. Med., 2:55-78 (2010)),and T stem memory cells (Gattinoni, et al., Nat. Med., 17:1290-1297(2012)) may all have certain advantages in certain applications due, forexample, to their high replicative capacity. Tumor InfiltratingLymphocytes (TILs) also have certain advantages due to their antigenspecificity and may be used in the delivery strategies disclosed herein.

Although sometime referred to as CAR cells, CAR immune, cells, and CARTcells (or CAR T cells), it will be appreciated that the CAR and otherdelivery strategies disclosed herein can also be carried out in othercell types, particularly different types of immune cells, includingthose discussed herein (e.g., lymphocytes, Natural Killer Cells,dendritic cells, B cells, antigen presenting cells, macrophage, etc.)and described elsewhere (see, e.g., Barrett, et al., Annu Rev Med., 65:333-347 (2014)).

4. Cancer Cells and Tumors

In some embodiments, the target cells are cancer cells. In suchembodiments, methods of treatment are provided that may be useful in thecontext of cancer, including tumor therapy. The Examples below mayindicate that DNA cargo may be delivered more generally to multipletissues and not restricted to tumors, while RNA delivery may be moreselective for tumor tissue. Thus, in some embodiments, when cancer cellsare the target cells, the cargo may be composed of RNA (e.g., RNAalone).

Cargos that may be delivered to cancer cells include, but are notlimited to, constructs for the expression of one or more pro-apoptoticfactors, immunogenic factors, or tumor suppressors; gene editingcompositions, inhibitory nucleic acids that target oncogenes; as well asother strategies discussed herein and elsewhere. In some embodiments,the cargo is mRNA that encodes a pro-apoptotic factor, or immunogenicfactor that increases and immune response against the cells. In otherembodiments, the cargo is siRNA the reduces expression of an oncogene orother cancer-causing transcript.

In a mature animal, a balance usually is maintained between cell renewaland cell death in most organs and tissues. The various types of maturecells in the body have a given life span; as these cells die, new cellsare generated by the proliferation and differentiation of various typesof stem cells. Under normal circumstances, the production of new cellsis so regulated that the numbers of any particular type of cell remainconstant. Occasionally, though, cells arise that are no longerresponsive to normal growth-control mechanisms. These cells give rise toclones of cells that can expand to a considerable size, producing atumor or neoplasm. A tumor that is not capable of indefinite growth anddoes not invade the healthy surrounding tissue extensively is benign. Atumor that continues to grow and becomes progressively invasive ismalignant. The term cancer refers specifically to a malignant tumor. Inaddition to uncontrolled growth, malignant tumors exhibit metastasis. Inthis process, small clusters of cancerous cells dislodge from a tumor,invade the blood or lymphatic vessels, and are carried to other tissues,where they continue to proliferate. In this way a primary tumor at onesite can give rise to a secondary tumor at another site.

The compositions and methods described herein may be useful for treatingsubjects having benign or malignant tumors by delaying or inhibiting thegrowth of a tumor in a subject, reducing the growth or size of thetumor, inhibiting or reducing metastasis of the tumor, and/or inhibitingor reducing symptoms associated with tumor development or growth.

Malignant tumors which may be treated are classified herein according tothe embryonic origin of the tissue from which the tumor is derived.Carcinomas are tumors arising from endodermal or ectodermal tissues suchas skin or the epithelial lining of internal organs and glands. Thedisclosed compositions are particularly effective in treatingcarcinomas. Sarcomas, which arise less frequently, are derived frommesodermal connective tissues such as bone, fat, and cartilage. Theleukemias and lymphomas are malignant tumors of hematopoietic cells ofthe bone marrow. Leukemias proliferate as single cells, whereaslymphomas tend to grow as tumor masses. Malignant tumors may show up atnumerous organs or tissues of the body to establish a cancer.

The types of cancer that can be treated with the provided compositionsand methods include, but are not limited to, cancers such as vascularcancer such as multiple myeloma, adenocarcinomas and sarcomas, of bone,bladder, brain, breast, cervical, colo-rectal, esophageal, kidney,liver, lung, nasopharangeal, pancreatic, prostate, skin, stomach, anduterine. In some embodiments, the disclosed compositions are used totreat multiple cancer types concurrently. The compositions can also beused to treat metastases or tumors at multiple locations.

The disclosed compositions and methods can be further understood throughthe following numbered paragraphs.

1. A composition comprising or consisting of

(a) a 3E10 monoclonal antibody, cell-penetrating fragment thereof; amonovalent, divalent, or multivalent single chain variable fragment(scFv); or a diabody; or humanized form or variant thereof, and

(b) a nucleic acid cargo comprising a nucleic acid encoding apolypeptide, a functional nucleic acid, a nucleic acid encoding afunctional nucleic acid, or a combination thereof.

2. The composition of paragraph 1, wherein (a) comprises:

(i) the CDRs of any one of SEQ ID NO:1-6, 12, 13, 46-48, or 50-52 incombination with the CDRs of any one of SEQ ID NO:7-11, 14, or 53-58;

(ii) first, second, and third heavy chain CDRs selected from any of SEQID NOS:15-23, 42, or 43 in combination with first, second and thirdlight chain CDRs selected from any of SEQ ID NOS:24-30, 44, or 45;

(iii) a humanized form of (i) or (ii);

(iv) a heavy chain comprising an amino acid sequence comprising at least85% sequence identity to any one of SEQ ID NO:1 or 2 in combination witha light chain comprising an amino acid sequence comprising at least 85%sequence identity to SEQ ID NO:7 or 8;

(v) a humanized form or (iv); or

(vi) a heavy chain comprising an amino acid sequence comprising at least85% sequence identity to any one of SEQ ID NO:3-6, 46-48, or 50-52 incombination with a light chain comprising an amino acid sequencecomprising at least 85% sequence identity to SEQ ID NO:9-11 or 53-58.

3. The composition of paragraphs 1 or 2, wherein (a) comprises the sameor different epitope specificity as monoclonal antibody 3E10, producedby ATCC Accession No. PTA 2439 hybridoma.

4. The composition of any one of paragraphs 1-3, wherein (a) is arecombinant antibody having the paratope of monoclonal antibody 3E10.

5. A composition comprising

(a) a binding protein comprising

-   -   (i) the CDRs of any one of SEQ ID NO:1-6, 12, 13, 46-48, or        50-52 in combination with the CDRs of any one of SEQ ID NO:7-11,        14, or 53-58;    -   (ii) first, second, and third heavy chain CDRs selected from SEQ        ID NOS:15-23, 42, or 43 in combination with first, second and        third light chain CDRs selected from SEQ ID NOS:24-30, 44, or        45;    -   (iii) a humanized form of (i) or (ii);    -   (iv) a heavy chain comprising an amino acid sequence comprising        at least 85% sequence identity to any one of SEQ ID NO:1 or 2 in        combination with a light chain comprising an amino acid sequence        comprising at least 85% sequence identity to SEQ ID NO:7 or 8;    -   (v) a humanized form or (iv); or    -   (vi) a heavy chain comprising an amino acid sequence comprising        at least 85% sequence identity to any one of SEQ ID NO:3-6,        46-48, or 50-52 in combination with a light chain comprising an        amino acid sequence comprising at least 85% sequence identity to        SEQ ID NO:9-11 or 53-58, and

(b) a nucleic acid cargo comprising a nucleic acid encoding apolypeptide, a functional nucleic acid, a nucleic acid encoding afunctional nucleic acid, or a combination thereof.

6. The composition of any one of paragraphs 1-5, wherein (a) isbispecific.

7. The composition of paragraph 6, wherein (a) targets a cell type ofinterest.

8. The composition of any one of paragraphs 1-7, wherein (a) and (b) arenon-covalently linked.

9. The composition of any one of paragraphs 1-8, wherein (a) and (b) arein a complex.

10. The composition of any one of paragraphs 1-9 wherein (b) comprisesDNA, RNA, PNA or other modified nucleic acids, or nucleic acid analogs,or a combination thereof.

11. The composition of any one of paragraphs 1-10, wherein (b) comprisesmRNA.

12. The composition of any one of paragraphs 1-11, wherein (b) comprisesa vector.

13. The composition of paragraph 12, wherein the vector comprises anucleic acid sequence encoding a polypeptide of interest operably linkedto expression control sequence.

14. The composition of paragraph 13, wherein the vector is a plasmid.

15. The composition of any one of paragraphs 1-14, wherein (b) comprisesa nucleic acid encoding a Cas endonuclease, a gRNA, or a combinationthereof.

16. The composition of any one of paragraphs 1-15, wherein (b) comprisesa nucleic acid encoding a chimeric antigen receptor polypeptide.

17. The composition of any one of paragraphs 1-16, wherein (b) comprisesa functional nucleic acid.

18. The composition of any one of paragraphs 1-17, wherein (b) comprisesa nucleic acid encoding a functional nucleic acid.

19. The composition of paragraphs 17 or 18, wherein the functionalnucleic acid is antisense molecules, siRNA, miRNA, aptamers, ribozymes,RNAi, or external guide sequences.

20. The composition of any one of paragraphs 1-19, wherein (b) comprisesa plurality of a single nucleic acid molecules.

21. The composition of any one of paragraphs 1-19, wherein (b) comprisesa plurality of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleicacid molecules.

22. The composition of any one of paragraphs 1-21, wherein (b) comprisesor consists of nucleic acid molecules between about 1 and 25,000nucleobases in length.

23. The composition of any one of paragraphs 1-22, wherein (b) comprisesor consists of single stranded nucleic acids, double stranded nucleicacids, or a combination thereof.

24. The composition of any one of paragraphs 1-23, further comprisingcarrier DNA.

25. The composition of paragraph 24, wherein the carrier DNA isnon-coding DNA.

26. The composition of paragraphs 24 or 25, wherein (b) is composed ofRNA.

27. A pharmaceutical composition comprising the composition of any oneof paragraphs 1-26 and a pharmaceutically acceptable excipient.

28. The composition of paragraph 27 further comprising polymericnanoparticles encapsulating a complex of (a) and (b).

29. The composition of paragraph 28, wherein a targeting moiety, a cellpenetrating peptide, or a combination thereof is associated with,linked, conjugated, or otherwise attached directly or indirectly to thenanoparticle.

30. A method of delivering a nucleic acid cargo to a cell comprisingcontacting the cell with an effective amount of the composition of anyone of paragraphs 1-29.

31. The method of paragraph 30, wherein the contacting occurs ex vivo.

32. The method of paragraph 31, wherein the cells are hematopoietic stemcells, or T cells.

33. The method of any one of paragraphs 30-32, further comprisingadministering the cells to a subject in need thereof.

34. The method of paragraph 33, wherein the cells are administered tothe subject in an effective amount to treat one or more symptoms of adisease or disorder.

35. The method of paragraph 30 wherein the contacting occurs in vivofollowing administration to a subject in need thereof.

36. The method of any one of paragraphs 33-35, wherein the subject has adisease or disorder.

37. The method of paragraph 36, wherein the disease or disorder is agenetic disorder, cancer, or an infection or infectious disease.

38. The method of paragraphs 36 or 37, wherein (b) is delivered intocells of the subject in an effective amount to reduce one or moresymptoms of the disease or disorder in the subject.

39. A method of making the composition of any one of paragraphs 1-29comprising incubating and/or mixing of (a) and (b) for an effectiveamount of time and at a suitable temperature to form complexes of (a)and (b), prior to contact with cells.

40. A method of making the composition of any one of paragraphs 1-29,comprising incubating and/or mixing of (a) and (b) for between about 1min and about 30 min, about 10 min and about 20 min, or about 15 min,optionally at room temperature or 37 degrees Celsius.

41. A composition or method of any one of paragraphs 1-40 wherein 3E10monoclonal antibody, cell-penetrating fragment thereof; a monovalent,divalent, or multivalent single chain variable fragment (scFv); or adiabody; or humanized form or variant thereof comprising the nucleicacid binding pocket of SEQ ID NOS:92 or 93, or a variant thereof withsame or improved ability to bind to a nucleic acid.

42. A composition or method of any one of paragraphs 1-41 wherein theamino acid residue corresponding with D31 or N31 of a heavy chain aminoacid sequence or a CDR thereof is substituted with R.

43. A composition or method of any one of paragraphs 1-42 wherein theamino acid residue corresponding with D31 or N31 of a heavy chain aminoacid sequence or a CDR thereof is substituted with L.

44. A binding protein comprising

-   -   (i) a variant of CDRs of any one of SEQ ID NO:1-6, 12, 13,        46-48, or 50-52 in combination with the CDRs of any one of SEQ        ID NO:7-11, 14, or 53-58;    -   (ii) a variant of the first heavy chain CDR, in combination with        the second, and third heavy chain CDRs selected from SEQ ID        NOS:15-23, 42, or 43 in combination with first, second and third        light chain CDRs selected from SEQ ID NOS:24-30, 44, or 45;    -   (iii) a humanized form of (i) or (ii);    -   (iv) a heavy chain comprising an amino acid sequence comprising        at least 85% sequence identity to any one of SEQ ID NO:1 or 2 in        combination with a light chain comprising an amino acid sequence        comprising at least 85% sequence identity to SEQ ID NO:7 or 8;    -   (v) a humanized form or (iv); or    -   (vi) a heavy chain comprising an amino acid sequence comprising        at least 85% sequence identity to any one of SEQ ID NO:3-6,        46-48, or 50-52 in combination with a light chain comprising an        amino acid sequence comprising at least 85% sequence identity to        SEQ ID NO:9-11 or 53-58,    -   wherein the amino acid residue corresponding with D31 or N31 is        substituted with R or L.

45. The binding protein of paragraph 44, comprising the nucleic acidbinding pocket of SEQ ID NOS:92 or 93, or a variant thereof with same orimproved ability to bind to a nucleic acid.

EXAMPLES

With respect to the experiments below, standard 3E10 sequence was usedexcept wherein noted to be the D31N variant (e.g., Example 4). Bothstandard 3E10 and the D31N variant were used as full length antibodies.

Example 1: 3E10 Increases Cellular Uptake of PNA after 1 Hour Materialsand Methods

PNA alone (1 nmole) (MW=9984.39; 29 nucleotides in length), or PNAcomplexed with 3E10 (0.75 mg), was mixed at room temperature for 5minutes. 200,000 K562 cells were then added to the suspension of 3E10,or PNA alone, in serum free media. Additional serum free media was addedto a final volume of 500 ul. Following incubation with cells at 37° C.for 1 hr, the cells were centrifuged and washed three times with PBSprior to analysis by flow cytometry. The PNA was labeled by attachmentto the fluorescent dye, tetramethylrhodamine (TAMRA).

Results

The results are illustrated in flow cytometry dot plots (FIG. 1A-1C). %uptake was quantified (FIG. 1D).

The results show increased uptake of PNA when mixed with 3E10.

Example 2: 3E10 Increases Cellular Uptake of PNA after 24 HoursMaterials and Methods

PNA alone (1 nmole) (MW=9984.39; 29 nucleotides in length), or PNAcomplexed with 3E10 (0.75 mg), was mixed at room temperature for 5minutes. 200,000 K562 cells were then added to the suspension of 3E10,or PNA alone, in serum free media. Additional serum free media was addedto a final volume of 500 ul. Following incubation with cells at 37° C.for 24 hrs, the cells were centrifuged and washed three times with PBSprior to analysis by flow cytometry.

20,000 U2OS cells were seeded in 8-well chamber slides and allowed toadhere for 24 hours. Cells were subsequently treated with PNA alone (1nmole), or PNA complexed with 3E10 (10 uM). Following incubation at 37°C. for 24 hrs, PNA or PNA mixed with 3E10 was washed with PBS prior tofixation and nuclear staining PNA uptake was subsequently quantified byflow cytometry and imaged using fluorescent microscopy. The PNA waslabeled by attachment to the fluorescent dye, tetramethylrhodamine(TAMRA).

Results

The results are illustrated in flow cytometry dot plots (FIG. 2A-2C). %uptake was quantified (FIG. 2D).

The results show increased uptake of PNA when mixed with 3E10.

Fluorescent microscopy showed co-localization of nuclear DNA (DAPI inblue) and PNA (Tamra in red) evident by the production of a distinctpink hue.

Example 3: 3E10 Increases Cellular Uptake of siRNA after 24 HoursMaterials and Methods

Labeled siRNA (via attachment to fluorescein amidite, FAM) (1 nmole), orsiRNA complexed with 3E10 (0.75 mg), was mixed at room temperature for 5minutes. 200,000 K562 cells were then added to the suspension of 3E10,or siRNA alone, in serum free media. Additional serum free media wasadded to a final volume of 500 ul. Following incubation with cells at37° C. for 24 hrs, the cells were centrifuged and washed three timeswith PBS prior to analysis by flow cytometry.

Results

The results are illustrated in flow cytometry dot plots (FIG. 3A-3C). %uptake was quantified (FIG. 3D).

The results show increased cell uptake of siRNA when mixed with 3E10.

Example 4: 3E10 Increases Cellular Uptake of mRNA after 24 HoursMaterials and Methods

Labeled mRNA (by attachment to cyanine 5, Cy5) (2 ug) alone or labeledmRNA complexed with 3E10 (2.5, 5, and 10 uM), were mixed at roomtemperature for 5 minutes. The suspensions of 3E10 plus mRNA, or mRNAalone, were added to 200,000 K562 cells in serum free media. Additionalserum free media was added to a final volume of 500 ul. Followingincubation with cells at 37° C. for 24 hrs, the cells were centrifugedand washed three times with PBS prior to analysis by flow cytometry.

Results

The results are illustrated in flow cytometry dot plots (FIG. 4A-4H). %uptake was quantified (FIG. 4I).

The results show increased uptake of mRNA when mixed with 3E10. Notethat delivery of mRNA by the D31N variant of 3E10 resulted in thehighest levels of mRNA cell uptake.

Fluorescent microscopy showed functional GFP expression in U2OS cellsafter translation of the same Cy5 labeled mRNA, which encodes for agreen fluorescent protein (GFP) reporter.

Example 5: 3E10 Increases Cellular Uptake of mRNA after 1 Hours

Materials and Methods

Labeled mRNA (Cy5) (2 ug) or labeled mRNA complexed with the D31Nvariant of 3E10 (0.1-10 uM) were mixed at room temperature for 5minutes. The suspensions of 3E10 plus mRNA, or mRNA alone, were added to200,000 K562 cells in serum free media. Additional serum free media wasadded to a final volume of 500 ul. Following incubation with cells at37° C. for 1 hr, the cells were centrifuged and washed three times withPBS prior to analysis by flow cytometry.

Results

The results are illustrated in flow cytometry dot plots (FIG. 5A-5H). %uptake was quantified (FIG. 5I).

Example 6: 3E10 Increases Cellular Uptake of Plasmid DNA Materials andMethods

GFP reporter plasmid DNA (250 ug) was complexed with 3E10 (10 uM) atroom temperature for 5 minutes. The suspension of 3E10 plus plasmid DNA,or plasmid DNA alone, were added to 200,000 K562 cells in serum freemedia. Additional serum free media was added to a final volume of 500ul. Following incubation with cells at 37° C. for 24 hrs, the cells werecentrifuged and washed three times with PBS. 72 hours after the initialtreatment, cells were imaged and analyzed for GFP expression.

Results

Results indicate that GFP plasmid was robustly taken up by cells when3E10 was combined with the plasmid DNA, as measured by greenfluorescence, indicating uptake and functional expression of the GFPconstruct. No uptake or green fluorescence was seen when plasmid DNAalone was used. (FIG. 6 ).

Example 7: 3E10 Mediates mRNA Delivery In Vivo Materials and Methods

10 ug of mRNA encoding GFP was mixed with 0.1 mg of 3E10 for 15 minutesat room temperature. mRNA complexed to 3E10 was injected systemically toBALB/c mice bearing EMT6 flank tumors measuring 100 mm³. 20 hours aftertreatment, tumors were harvested and analyzed for mRNA expression (GFP)using IVIS imaging.

Results

3E10-mediated delivery of mRNA resulted in significantly higher levelsof GFP expression in the tumor compared to freely injected mRNA, whichdid not yield any GFP expression in the tumor. There was no detectableexpression of GFP in any of the normal tissues examined with eithertreatment, including liver, spleen, heart, and kidney. The resultsindicate robust delivery of mRNA into tumors, with functionaltranslation and expression.

Example 8: 3E10 Mediates siRNA Delivery In Vivo Materials and Methods

40 ug of fluorescently labeled siRNA was mixed with increased doses of3E10 (0.25, 0.5, and 1 mg) for 15 minutes at room temperature. siRNAcomplexed to 3E10 was injected systemically to BALB/c mice bearing EMT6flank tumors measuring 100 mm³. 20 hours after treatment, tumors wereharvested and analyzed for siRNA delivery using IVIS imaging.

40 ug of fluorescently labeled siRNA was mixed with 1 mg 3E10 or 0.1 mgof the D31N variant of 3E10 for 15 minutes at room temperature. siRNAcomplexed to 3E10 was injected systemically to BALB/c mice bearing EMT6flank tumors measuring 100 mm³. 20 hours after treatment, tumors wereharvested and analyzed for siRNA delivery using IVIS imaging.

Results

As shown in FIG. 7A, increasing doses of 3E10 result in higheraccumulation of siRNA in tumors.

As shown in FIG. 7B, a tenfold lower dose of D31N 3E10 resulted insimilar levels of siRNA delivery as 3E10.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Example 9: Carrier DNA Enhances mRNA to Non-Tumor Tissue Materials andMethods

2 ug of fluorescently labeled mRNA was mixed with 20 ug of 3E10-D31Nwith or without carrier DNA (5 ug) for 15 minutes at room temperature.mRNA complexed to 3E10 was injected to fetuses at E15.5. 24-48 hoursafter treatment, fetuses were harvested and analyzed for mRNA deliveryusing IVIS imaging.

Results

Without carrier DNA, 3E10-D31N complexed to mRNA was rapidly clearedfrom fetuses at 24 hours. The addition of carrier DNA, however, resultedin detectable mRNA signal in multiple tissues of the fetus at 48 hours.

The Examples above may indicate that DNA cargo delivery may be moregeneral to multiple tissues and not restricted to tumors, while RNAdelivery may be more selective for tumor tissue.

Example 10: 3E10 (D31N) Complexed with mRNA and Carrier DNA Results inSustained Levels Protein Expression Materials and Methods

10 ug of luciferase mRNA and 10 ug of single stranded carrier DNA (60nts) was mixed with 100 ug of 3E10 (WT) or 3E10 (D31N) for 15 minutes atroom temperature. mRNA complexed to 3E10 was injected intramuscularly(IM) in the right quadricep of each mouse. Luciferase expression wasmonitored over 6 days.

Results

As seen in FIG. 8 , administration of 3E10 (D31N) complexed with mRNAand carrier DNA resulted in sustained levels of luciferase expression,while 3E10 (WT) complexed to mRNA and carrier DNA failed to produce anyappreciable signal above background.

Example 11: Distribution of IV Injected 3E10 In Vivo

Distribution of IV injected 3E10 to muscle was investigated. Mice wereinjected intravenously with 200 μg of 3E10, WT or D31N, labeled withVivoTag680 (Perkin Elmer). Four hours after injection, muscle washarvested and imaged by IVIS (Perkin Elmer) (FIGS. 9A and 9B).Quantification of IVIS image demonstrates that 3E10-D31N achieves higherdistribution to muscle when compared to 3E10-WT (FIG. 9C).

Dose-dependent biodistribution of 3E10-D31N to tissues was investigated.Mice were injected intravenously with 100 μg or 200 μg of 3E10-D31Nlabeled with VivoTag680 (Perkin Elmer). 24 hours after injection,tissues were harvested and imaged by IVIS (Perkin Elmer). Quantificationof tissue distribution demonstrated a dose-dependent, two-fold increasein muscle accumulation without a commensurate increase in multipletissues including liver (FIG. 10 ).

Distribution of 3E10 to tumors. Mice bearing flank syngeneic colontumors (CT26) were injected intravenously with 200 μg of 3E10, WT orD31N, labeled with VivoTag680 (Perkin Elmer). 24 hours after injection,tumors were harvested and imaged by IVIS (Perkin Elmer) (FIG. 11A-11B).Quantification of tumor distribution demonstrated that 3E10-D31N hadhigher accumulation in tumors when compared to 3E10-WT (FIG. 11C).

Distribution of ssDNA non-covalently associated with 3E10 wasinvestigated. Mice bearing flank syngeneic colon tumors (CT26) wereinjected intravenously with 200 ug of 3E10, WT or D31N, mixed with 40 ugof labeled ssDNA (IR680). 24 hours after injection, tumors wereharvested and imaged by IVIS (Perkin Elmer) (FIG. 12A-12C).Quantification of tissue distribution demonstrated that delivery ofssDNA by 3E10-D31N resulted in higher tumor accumulation when comparedto 3E10-WT (FIG. 12D).

Example 12: 3E10-Mediates Delivery of RIG-I Ligand, and Stimulation ofRIG-I Activity Materials and Methods

RIG-I reporter cells (HEK-Lucia RIG-I, Invivogen) were seeded at 50,000cells per well and treated with RIG-I ligands (lug) or ligands complexedto 3E10-D31N (20 ug). This assay uses a cell line with a luciferasereporter that is activated when there is induction of interferons.

Results

In all cases, RIG-I ligands alone did not stimulate IFN-γ secretion.Delivery of RIG-ligands with 3E10-D31N, however, stimulated IFN-γsecretion above controls, with the highest secretion observed for poly(I:C), both low and high molecular weight (LMW and HMW).

Example 13: Molecular Modeling of 3E10 and Engineered Variants Thereof

WT HEAVY CHAIN scFv SEQUENCE (SEQ ID NO: 92)E VQLVESGGGL VKPGGSRKLS CAASGFTFSD YGMHWVRQAPEKGLEWVAYI SSGSSTIYYA DTVKGRFTIS RDNAKNTLFLQMTSLRSEDT AMYYCARRGL LLDYWGQGTT LTVS LIGHT CHAIN scFv SEQUENCE(SEQ ID NO: 93) D IVLTQSPASL AVSLGQRATI SCRASKSVST SSYSYMHWYQQKPGQPPKLL IKYASYLESG VPARFSGSGS GTDFTLNIHPVEEEDAATYY CQHSREFPWT FGGGTKLEIK RADAAPGGGG SGGGGSGGGGS

Molecular modeling of 3E10 (Pymol) revealed a putative Nucleic AcidBinding pocket (NAB1) (FIGS. 14A-14B). Mutation of aspartic acid atresidue 31 of CDR1 to asparagine increased the cationic charge of thisresidue and enhanced nucleic acid binding and delivery in vivo(3E10-D31N).

Mutation of aspartic acid at residue 31 of CDR1 to arginine (3E10-D31R),further expanded the cationic charge while mutation to lysine(3E10-D31K) changed charge orientation (FIG. 14A).

NAB1 amino acids predicted from molecular modeling have been underlinedin the heavy and light chain sequences above. FIG. 14B is anillustration showing molecular modeling of 3E10-scFv (Pymol) with NAB1amino acid residues illustrated with punctate dots.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1.-45. (canceled)
 46. A composition comprising a non-covalent complex of(a) a 3E10 antibody or antigen-binding fragment thereof, and (b) anmRNA.
 47. The composition according to claim 46, wherein the 3E10antibody or antigen-binding fragment thereof comprises a heavy chainvariable region (V_(H)) having an amino acid sequence that is at least95% identical to SEQ ID NO:2 and a light chain variable region (V_(L))having an amino acid sequence that is at least 95% identical to SEQ IDNO:7 or
 8. 48. The composition according to claim 47, wherein the 3E10antibody or antigen-binding fragment thereof, comprises V_(H)complementarity determining regions (CDRs) having the amino acidsequences of SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, and V_(L)CDRs having the amino acid sequences of SEQ ID NO:24, SEQ ID NO:25, andSEQ ID NO:26.
 49. The composition according to claim 47, wherein theV_(H) has the amino acid sequence of SEQ ID NO:2 and the V_(L) has theamino acid sequence of SEQ ID NO:7 or
 8. 50. The composition accordingto claim 46, wherein the 3E10 antibody or antigen-binding fragmentthereof comprises a V_(H) having an amino acid sequence that is at least95% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 3-6, 46-48, and 50-52 and a V_(L) having anamino acid sequence that is at least 95% identical to an amino acidsequence selected from the group consisting of SEQ ID NOs: 9-11 and53-58.
 51. The composition according to claim 50, wherein the 3E10antibody or antigen-binding fragment thereof, comprises V_(H)complementarity determining regions (CDRs) having the amino acidsequences of SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, and V_(L)CDRs having the amino acid sequences of SEQ ID NO:24, SEQ ID NO:25, andSEQ ID NO:26.
 52. The composition according to claim 50, wherein theV_(H) has an amino acid sequence selected from the group consisting ofSEQ ID NOs: 3-6, 46-48, and 50-52 and the V_(L) has an amino acidsequence selected from the group consisting of SEQ ID NOs: 9-11 and53-58.
 53. The composition according to claim 46, wherein the 3E10 orantigen-binding fragment thereof, comprises V_(H) complementaritydetermining regions (CDRs) having the amino acid sequences of SEQ IDNO:16, SEQ ID NO:17, and SEQ ID NO:18, and V_(L) CDRs having the aminoacid sequences of SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26.
 54. Thecomposition according to claim 46, wherein the 3E10 antibody orantigen-binding fragment thereof is a humanized antibody.
 55. Thecomposition according to claim 54, wherein the 3E10 antibody orantigen-binding fragment thereof, comprises V_(H) complementaritydetermining regions (CDRs) having the amino acid sequences of SEQ IDNO:16, SEQ ID NO:17, and SEQ ID NO:18, and V_(L) CDRs having the aminoacid sequences of SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26.
 56. Thecomposition according to claim 46, wherein the 3E10 antibody orantigen-binding fragment thereof is a monovalent, divalent, ormultivalent single chain variable fragment (scFv).
 57. The compositionaccording to claim 56, wherein the 3E10 or antigen-binding fragmentthereof, comprises V_(H) complementarity determining regions (CDRs)having the amino acid sequences of SEQ ID NO:16, SEQ ID NO:17, and SEQID NO:18, and V_(L) CDRs having the amino acid sequences of SEQ IDNO:24, SEQ ID NO:25, and SEQ ID NO:26.
 58. The composition of claim 46,wherein the 3E10 or antigen-binding fragment thereof is a bispecificantibody.
 59. The composition of claim 58, wherein the bispecificantibody comprises: a first 3E10 heavy chain or antigen-binding fragmentthereof, and a first 3E10 light chain or antigen binding fragmentthereof; and a second heavy chain, or antigen-binding fragment thereof,and a second light chain, or antigen binding fragment thereof, thatassociate to specifically bind a target cell-type, tissue, or organ. 60.The composition of claim 59, wherein: the first 3E10 heavy chain orantigen-binding fragment thereof, comprises V_(H) CDRs having the aminoacid sequences of SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18; and thefirst 3E10 light chain or antigen-binding fragment thereof, comprisesV_(L) CDRs having the amino acid sequences of NO:24, SEQ ID NO:25, andSEQ ID NO:26.
 61. The composition according to claim 46, wherein themRNA encodes a polypeptide ligand for a receptor of an immune cell. 62.The composition according to claim 61, wherein the polypeptide ligand isa cytokine.
 63. The composition according to claim 61, wherein thepolypeptide ligand stimulates the immune system of the subject.
 64. Thecomposition according to claim 46, wherein the mRNA encodes apolypeptide that is defective in a genetic disease.
 65. The compositionaccording to claim 64, wherein the mRNA encodes dystrophin.
 66. Thecomposition according to claim 64, wherein the mRNA encodes utrophin.67. The composition according to claim 46, wherein the mRNA encodes anantigen.
 68. The composition according to claim 67, wherein the antigenis a viral antigen.
 69. The composition according to claim 68, whereinthe viral antigen is a SARS-CoV-2 antigen.
 70. The composition accordingto claim 46, wherein the mRNA encodes a pro-apoptotic factor.
 71. Thecomposition according to claim 70, wherein the pro-apoptotic factor is aBH3 mimetic.
 72. The composition according to claim 46, wherein the mRNAencodes a tumor suppressor.
 73. The composition according to claim 46,further comprising a pharmaceutically acceptable excipient.